Antibodies

ABSTRACT

The present invention provides antibodies which bind to an epitope in the extracellular domain of human CC chemokine receptor 4 (CCR4) and which are capable of inhibiting the binding of macrophage-derived chemokine (MDC) and/or thymus and activation regulated chemokine (TARC) to CCR4. Also provided are inter alia immunoconjugates and compositions comprising such antibodies and methods and uses involving such antibodies, particularly in the medical and diagnostic fields.

This application is a continuation application of U.S. Ser. No.13/870,058, filed 25 Apr. 2013 which is a continuation of Ser. No.12/797,184, filed 9 Jun. 2010, now U.S. Pat. No. 8,461,304, issued 11Jun. 2013 which claims priority from U.S. provisional application No.61/185,448, filed 9 Jun. 2009, U.S. provisional application No.61/302,768, filed 9 Feb. 2010 and GB Application No. 0909906.0, filed 9Jun. 2009. These prior applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of antibodies,CCR4 biology and related therapies. More particularly, it providesantibodies that bind to CCR4. Such anti-CCR4 antibodies have diagnosticand therapeutic uses in diseases and conditions associated with CCR4,such as in imaging tumour blood vessels, treating cancer and treatingviral and other infections, and inflammatory and immune diseases. Theantibody-based compositions and methods of the invention also extend tothe use of immunoconjugates and other therapeutic combinations, kits andmethods.

BACKGROUND

With more than 800 members, G-protein-coupled receptors (GPCRs)represent the largest family of cell surface molecules involved insignal transmission, accounting for >2% of the total genes encoded byhuman genome. Members of the GPCR superfamily share a common membranetopology: an extracellular N-terminus, an intracellular C-terminus andseven transmembrane (TM) helices, which are connected by threeintracellular loops and three extracellular loops. On the basis of theirshared topological structure, GPCRs are also referred to as seventransmembrane (7TM) receptors. These receptors control key physiologicalfunctions, including neurotransmission, hormone and enzyme release fromendocrine and exocrine glands, immune responses, cardiac- andsmooth-muscle contraction and blood pressure regulation. Theirdysfunction contributes to some of the most prevalent human diseases.Emerging experimental and clinical data indicate that GPCRs have acrucial role in cancer progression and metastasis. Hence, there is thepossibility that some GPCRs may be suitable targets for anti-cancerdrugs.

Chemokines play an important role inter alia in immune and inflammatoryresponses in various diseases and disorders, including cancer, viralinfections, asthma and allergic diseases, as well as autoimmunepathologies such as rheumatoid arthritis and atherosclerosis. Thesesmall, secreted molecules are a growing superfamily of 8-14 kDa proteinscharacterised by a conserved four cysteine motif.

Studies have demonstrated that the actions of chemokines are mediated bysubfamilies of G protein-coupled receptors, among which is the receptordesignated chemokine (C-C motif) receptor 4, or CC chemokine receptor 4(CCR4). Specific ligands for CCR4 include the chemokines thymus andactivation-regulated chemokine (TARC) (also known as CCL17) andmacrophage-derived chemokine (MDC) (also known as CCL22). CCR4 may alsobind to RANTES, MCP-1 and MIP-1alpha, and CCR4 signalling in response tothese ligands has also been reported.

CCR4 is believed to be important inter alia in the function of T cellchemotaxis and the migration of phagocytic cells to sites ofinflammation. CCR4 is preferentially expressed on T-helper cell type 2(Th2) cells and regulatory T (Treg) cells, whereas only limitedexpression on other healthy cells or tissues occurs.

Tumour cells, in particular adult T cell leukaemia/lymphoma cells, maybe positive for CCR4. Expression of CCR4 by tumour cells is associatedwith skin involvement. Certain T-cell malignancies typically are locatedto the skin. For example, CCR4 is found at high levels in cutaneous Tcell lymphoma lesions. More recently, CCR4 has also been found to beexpressed by certain solid tumours (WO2009/037454). CCR4 expression isbelieved to be an early event in carcinogenesis of solid tumours,particularly cancer of the cervix, oesophagus, kidney, brain, breast,ovary, prostate, stomach and pancreas. Thus, both haematological andnon-haematological cancer cells may express CCR4. Consequently, thesecancers maybe diagnosed, monitored and treated using anti-CCR4antibodies.

In addition, CCR4 has an important role in normal and tumour immunity. Asignificant fraction of CD4⁺ CD25⁺ regulatory T-cells (Tregs) arepositive for CCR4 (Baatar et al, 2007b). These Tregs suppress immuneresponses through a variety of mechanisms, and it has been shown thatthey can inhibit tumour-specific immunity. Increased numbers of Tregsinfiltrating the stroma, the tumour itself, or draining lymphnodes,correlate with worsened outcome in a variety of cancers. Studies inmouse model show that reducing Treg activity leads to increasedendogenous anti-tumour immunity and increased efficacy of anti-tumourinventions by the immune system. Consequently, inhibiting Treg functionis a promising strategy in immunotherapy of tumours. The inhibition canbe achieved by killing the Tregs (depletion), interfering with theirsuppressor functions, changing their trafficking pattern or changingtheir differentiation.

In a subset of patients with CCR4+ T-cell leukaemia/lymphoma, the tumourcells themselves function as Treg cells, contributing to tumour survivalin the face of host antitumour immune responses. In other types ofcancers, MDC and TARC are produced by tumour cells and the tumourmicroenvironment and attract CCR4+ Treg cells to the tumour, where theycreate a favourable environment for tumour escape from the host immuneresponses. A higher frequency of Tregs in peripheral blood of patientswith following cancers has been reported: Breast cancer, Colorectalcancer, Oesophageal cancer, Gastric cancer, Hepatocellular carcinoma,Lung cancer, Melanoma, Ovarian cancer and Pancreatic cancer. Treg cellshave been reported to create a favourable environment for tumours.Hence, blocking the interaction between CCR4 and its ligands such as MDCcould be useful in the treatment or prevention of cancers, especiallythe cancers listed above. It has been reported that in a SCID mousemodel, antibody to human MDC/CCL22 was able to block infiltration ofhuman Treg cells into transplanted human ovarian tumours. It is believedthat the Treg cells present in human solid tumours prevent immuneeffector responses developing which could contribute to the slowing oftumour growth and metastasis. Thus, killing of Treg cells in the tumourmass, and/or prevention of migration of Treg cells to the tumour sitesby using a neutralising MAb (monoclonal antibody) directed against CCR4may result in enhanced immune responses towards solid tumours, and actas an adjunct to conventional cytotoxic or anti-hormonal therapies.

Cancer causes about 13% of all human deaths. According to the AmericanCancer Society, 7.6 million people died from cancer in the world during2007, so there remains a strong and urgent need for further anti-cancertherapeutics.

CCR4 has been shown to play a role in inflammation and immune disorders.Th2 cells and basophils are key cells in the allergic response in thelung and skin. There are a number of reports which describe the presenceof CCR4-expressing T cells and concomitant expression of CCR4 ligands(MDC, TARC) on airway epithelial cells in bronchial biopsies inallergen-challenged asthmatics (Panina-Bordignon et al, 2001). CCR4+ Tcells are also found in increased numbers in patients with atopicdermatitis, with a marked reduction of CCR4+ T cells observed when thedisease improved. Using a humanized SCID mouse model of asthma, it wasshown that blockade of CCR4 with antibodies prior to allergen challengereduced allergic airway inflammation, as well as the levels of Th2cytokines in the lungs. Depletion of CCR4+ T cells via lung delivery ofa blocking antibody may be a suitable treatment option for asthmaticpatients. Targeted delivery of a CCR4 blocking antibody to the skin mayalso be an attractive treatment for atopic dermatitis.

In allergic asthma, the presence of high levels of allergen-specific IgEis a reflection of an aberrant Th2 cell immune response to commonlyinhaled environmental allergens. Asthma is characterized by infiltrationof Th2 lymphocytes and eosinophils and by the production of Th2chemokines. Allergens are presented to T cells by dendritic cells (DCs)that continuously sample incoming foreign antigens. Upon properactivation by DCs, allergen-specific lymphocytes that are present indiseased airways produce Th2 cytokines interleukin (IL)-4, IL-5 andIL-13 that furthermore control leukocyte extravasation, goblet cellhyperplasia and bronchial hyper-reactivity (BHR). TARC and MDC producedby DCs induce the selective migration of Th2 cells but not Th1 cellsthrough triggering CCR4 (Perros et al, 2009). It was shown in murinemodels of asthma, that treatment with anti-TARC antibodies reduced thenumber of CD4+ T cells and eosinophils in bronchoalveolar lavage (BAL)fluid, the production of Th2 cytokines and airway hyper-responsivenessafter allergen challenge (Kawasaki et al, 2001). In contrast,CCR4-deficient mice showed no protection against airway inflammation andBHR (Chvatchko et al, 2000). Using a humanized SCID mouse model ofasthma, it was shown that blockade of CCR4 with antibodies prior toallergen challenge reduced allergic airway inflammation as well as thelevels of Th2 cytokines in the lungs (Perros et al, 2009). These dataindicate that CCR4 blockade is a feasible strategy for inhibitingallergic inflammation in humans.

Treg cells may suppress dendritic cells (DCs), thereby facilitating thedevelopment and progression of diseases, particularly infectiousdiseases and cancer. Anti-CCR4 antibodies able to block the suppressionof dendritic cells by Treg cells may therefore be useful as adjuvants invaccines, particularly as adjuvants in tumour vaccination or vaccinationagainst infectious disease. Thus, an anti-CCR4 antibody may enhance thetherapeutic effect of a vaccine, particularly enhancing thevaccine-induced immune response.

CCR4 binding compounds have been reported to show efficacy in murineallergic inflammation (Purandare et al, 2007, Burdi et al. 2007). It hasbeen reported that a CCR4-binding compound has reasonable potency invivo, as CCR4 dependent recruitment of leucocytes to the peritoneuminduced by TARC was inhibited by almost 90%. Yokoyama and colleaguespresented a quinazoline derivative targeting CCR4 which proved in vivoto be effective in reducing hypersensitivity reactions in a mouse model(Yokoyama et al, 2008b); a derivative of this compound proved to beeffective in a similar in vivo mouse model upon oral administration(Yokoyama et al, 2009). Recently, a group of scientists has identified anumber CCR4 antagonists using in silico modelling approach (Bayry et al,2008; Davies et al, 2009). By docking compounds to modelled CCR4, theauthors found molecules able to bind within the transmembrane region.Sixteen compounds inhibited CCR4-mediated migration of CCRF-CEM cells.When CCR4 antagonists were tested for their adjuvant function in vivowith Mycobacterium tuberculosis and hepatitis B vaccines, enhancedimmunogenicity was observed for both cellular and humoral immuneresponses. The observed effect was ascribed to inhibition of Tregactivity (Bayry et al, 2008; Davies et al, 2009). The fact that asignificant fraction of Treg cells are CCR4-positive is well known inthe art (Baatar et al 2007b). The observed effect is believed to beuseful not only in the context of vaccination against infectiousdiseases (caused, for example by a virus, a bacterium, a mycobacteriumor a parasite such as protozoa), but also in the context of cancervaccines.

As the cause for the efficacy of these compounds as adjuvants is basedon inhibiting Tregs by blocking CCR4 mediated signaling, it is expectedthat antibodies binding to CCR4 in an antagonistic manner would work thesame way; the pharmacological advantages of antibodies compared to smallmolecule drugs are well known in the art. The only antibody against CCR4currently in development is to the best of our knowledge KW-0761 byKyowa-Hakko. However, this antibody is effective only by ADCC; it doesnot prevent ligand-mediated signalling through CCR4 receptor. Therefore,the antibodies described in this invention are expected to be clearlysuperior in their modulation of immune reactions via Tregs.

Another application where modulating Tregs is of clinical use is cancertreatment. Tregs can inhibit tumour-specific immunity and theirincreased numbers correlate with unfavourable prognosis and diseaseprogression in some cancers. Studies in mouse models demonstrate thatreducing Treg activity boosts endogenous anti-tumour immunity, andincreases the efficacy of active immune interventions. Consequently,inhibiting Treg function is a strategy worth considering in human cancerimmunotherapy (Curiel, 2008; Ruter et al, 2009). This inhibition can beachieved both by modulating Tregs, or by directly killing them.

Examples for this approaches are described in the art by compoundstargeting other surface of Treg like CD25. Daclizumab (Zenapax®; Roche))and basiliximab (Simulect®; Novartis) are anti-human CD25 antibodiesapproved for use in autoimmune diseases, transplantation and cancersincluding HTLV-1 induced adult T-cell lymphoma/leukaemia (Church, 2003).Denileukin diftitox (Ontak®, DAB389IL-2; Ligand Pharmaceuticals Inc.) isa recombinant protein fusing the active domain of diphtheria toxin tohuman IL-2. In 1998, FDA has approved it to treat cutaneous T cellleukaemia/lymphoma (Olsen et al, 2001), which usually are CD4+CD25+.Denileukin diftitox is targeted to the IL-2 receptor and is proposed tobe internalized through CD25 by endocytosis. There is also evidence thatDenileukin diftitox improves immunogenicity of a tumour vaccine inpatients with renal cell cancer (Dannull et al, 2005). In addition, arecent report showed that denileukin diftitox reduces Treg numbers andfunction in melanoma with improved melanoma-specific immunity (Mahnke etal, 2007)

Other molecules on Tregs which are targeted for cancer treatment orimproved cancer vaccine effects include GITR (glucocorticoid-inducedtumour necrosis factor receptor-related gene) (Levings et al, 2002),Toll-like receptors (TLR) are expressed ubiquitously on a variety ofmammalian cells, including human Tregs (Yang et al, 2004, Rutter et al,2009) and Cytotoxic T lymphocyte antigen-4 (CTLA-4; CD152) (Sutmuller etal. 2001). Currently, Phase II and III clinical trials of anti-CTLA-4monoclonal antibody therapy are being conducted in melanoma, and Phase Iand II trials are being conducted in other tumour types. Two humanmonoclonal antibodies are under investigation—ipilimumab (MDX-010;Bristol-Myers Squibb/Medarex) and tremelimumab (CP-675,206; Pfizer).

CCR4 has also been implicated inter alia in the following disorders:Adult T-cell leukemia/lymphoma, Peripheral T-cell lymphoma, CutaneousT-Cell Lymphoma (CTCL), unspecified Diffuse large B-cell lymphoma,Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, Epstein-Barrvirus (EBV) infection, Mycosis fungoides (a mature T-cell lymphoma),Sézary syndrome (a variant of mycosis fungoides), allergicbronchopulmonary aspergillosis (ABPA), Asthma, LPS-induced endotoxicshock, Allergic inflammation, T-cell mediated neurodegenerative diseasessuch as Multiple Sclerosis (MS), Autoimmune diseases such as Psoriasis,Castleman's disease and Rheumatoid arthritis (RA).

Due to their complex structures, GPCRs are considered as “difficulttargets” for raising specific antibodies. They can neither be easilypurified from the membrane fraction of lysed cells, nor be recombinantlyproduced in different expression systems as correctly folded solubleproteins. To the inventors' knowledge, to date all known attempts ofothers to generate anti-GPCR antibodies using phage display have provento be unsuccessful.

The difficulties associated with generating antibodies against GPCRs areset out in Hoogenboom et al., 1999. Furthermore, Sui et al. (2003)explain the difficulties associated with trying to obtain humanantibodies against the GPCR chemokine receptor CXCR4 and report thateven using the pathfinder method combined with step-back selection nospecific antibodies could be identified. Thus, in the field of GPCRs,the generation of specific antibodies remains a major challenge.

A murine monoclonal antibody called 1G1 which reacts with human CCR4 iscommercially available from BD Pharmingen. This antibody may be used forimmunoflurescent staining, but the antibody is not a neutralisingantibody.

A chimeric antibody to CCR4 designated KM2760 is disclosed in Ishida etal., 2006. The authors report that this antibody does not block thebinding of CCR4 to its ligands MDC or TARC.

The inventors have recognized that the identification of additionalantibodies that recognize CCR4 would be of benefit in expanding thenumber of therapeutic options. In particular, antibodies that block thebinding of CCR4 to one or more of its ligands would offer furthertherapeutic avenues.

The inventors have also recognized that the development of therapeuticagents for the treatment of humans that are better tolerated from animmunological perspective would be advantageous. In this regard, humanantibodies generally have at least three potential advantages for use inhuman therapy. First, the human immune system should not recognize theantibody as foreign. Second, the half-life in the human circulation willbe similar to naturally occurring human antibodies, allowing smaller andless frequent doses to be given. Third, because the effector portion ishuman, it will interact better with the other parts of the human immunesystem.

The art therefore still lacks anti-CCR4 antibodies that can be used inthe safe and effective treatment of patients having disorders in whichCCR4 is involved, including in long-term administration, and poseschallenges to the development of such antibodies.

In particular, there is a need for human antibodies to CCR4. Althoughhuman antibodies are generally recognized to display advantages, it isknown that the development of human antibodies that have high enoughaffinities and appropriate functional properties to make them candidatesfor successful human therapy is by no means straightforward. This iseven more so the case with GPCRs, due to their complex and transmembranenature.

There also remains a strong need for anti-CCR4 antibodies which canblock the binding between CCR4 and one or more of its ligands such asMDC and/or TARC.

DESCRIPTION OF THE INVENTION

The present invention overcomes certain limitations in the prior art byproviding new therapeutic compositions and methods for use in the safeand effective treatment of tumors, viral infections and other diseasesand conditions in which CCR4+ cells are involved such as inflammatory orimmune disorders. The invention is based on antibodies that bind toCCR4, preferably to an epitope within the extracellular domain of CCR4,particularly human antibodies. Such antibodies are effective in treatingtumors and viral infections and other diseases and conditions in whichCCR4+ cells are involved, such as inflammatory or immune disorders. Thecompositions and methods of the invention also extend to the use ofimmunoconjugates and combinations using this particular category ofantibodies.

A particular advantage of the present invention is that the antibodiesprovided inhibit binding of MDC and/or TARC to CCR4. This contrasts withthe leading antibodies in the clinical field, which do not inhibitbinding of MDC and/or TARC to CCR4.

The present inventors have prepared CCR4-specific antibodies that bindto CCR4. For example, the antibodies bind to CCR4+ cells, in particularHEK293T-cells transfected with CCR4, DT40-cells transfected with CCR4and CCRF-CEM cells which naturally express CCR4 (see Example 2).Importantly, the antibodies do not significantly bind to CCR4− cells,i.e. cells which do not express CCR4. Thus, the antibodies disclosedherein bind specifically to CCR4, making them suitable candidates fordiagnostics and therapy of the conditions discussed herein.

Amino acid and/or DNA sequences of preferred antibody molecules of theinvention which bind to an epitope in the extracellular domain of CCR4,their VH and VL domains including complementarity determining regions(CDRs), are set forth in the various SEQ ID NOs. listed herein.

Thus, the present invention provides an antibody which binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4.

Preferably, the antibody is isolated. Also preferably, the antibody ishuman. Preferably, the antibody binds to an epitope in the extracellulardomain of CCR4. The CCR4 is preferably human. Thus, any reference to“binding to CCR4” includes the preferred embodiment of “binding to anepitope in the extracellular domain of CCR4”.

Thus, the invention preferably provides an isolated human antibody whichbinds to an epitope in the extracellular domain of human CCR4 and whichis capable of inhibiting the binding of MDC to CCR4.

In one embodiment, the present invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4comprising a heavy chain CDR1 domain comprising the amino acid sequenceof SEQ ID NO: 1, 7, 19, 101, 125, 126, 135 or 136 or a sequencesubstantially homologous to any one of these sequences, SEQ ID NOs: 101,125, 126, 135 and 136 being especially preferred.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 comprises a heavy chain CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 2, 8, 20, 127 or 128 or a sequencesubstantially homologous to any one of these sequences, SEQ ID NOs: 8,127 and 128 being especially preferred.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 comprises a heavy chain CDR3 domain comprisingthe amino acid sequence of SEQ ID NO: 3, 9, 15 or 21 or a sequencesubstantially homologous to any one of these sequences, SEQ ID NO: 9being especially preferred.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 comprises a light chain CDR1 domain comprisingthe amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 108, 129 or 130 ora sequence substantially homologous to any one of these sequences, SEQID NOs: 10, 129 or 130 being especially preferred.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 comprises a light chain CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 110, 131 or 132 ora sequence substantially homologous to any one of these sequences SEQ IDNOs: 11, 131 and 132 being especially preferred.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 comprises a light chain CDR3 domain comprisingthe amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 112, 133, 134, 137or 138 or a sequence substantially homologous to any one of thesesequences, SEQ ID NOs: 12, 133, 134, 137 and 138 being especiallypreferred.

SEQ ID NOs: 133 and 134 contain amino acid Y at position 11, but it ispreferred that the amino acid at position 11 of these sequences is V.SEQ ID NOs: 137 and 138 correspond to SEQ ID NOs 133 and 134, exceptthat the amino acid at position 11 is V. Thus, in any embodimentsdisclosed herein, any reference to SEQ ID NO: 133 should be understoodto include a reference to SEQ ID NO: 137 and any reference to SEQ IDNO:134 should be understood to include a reference to SEQ ID NO: 138.

Thus, in certain embodiments, the invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising one or more heavy chain CDR domains, wherein the heavychain CDR domain is selected from the group consisting of:

(a) a heavy chain CDR1 domain comprising the amino acid sequence of SEQID NO: 1, 7, 19, 101, 125 or 126 or a sequence substantially homologousthereto;(b) a heavy chain CDR2 domain comprising the amino acid sequence of SEQID NO: 2, 8, 20, 127 or 128 or a sequence substantially homologousthereto; and(c) a heavy chain CDR3 domain comprising the amino acid sequence of SEQID NO: 3, 9, 15 or 21 or a sequence substantially homologous thereto.

The invention also provides, in certain embodiments an antibody thatbinds to binds to CCR4 and which is capable of inhibiting the binding ofMDC to CCR4 comprising one or more light chain CDR domains, wherein thelight chain CDR domain is selected from the group consisting of:

(a) a light chain CDR1 domain comprising the amino acid sequence of SEQID NO: 4, 10, 16, 22, 108, 129 or 130 or a sequence substantiallyhomologous thereto;(b) a light chain CDR2 domain comprising the amino acid sequence of SEQID NO: 5, 11, 17, 23, 110, 131 or 132 or a sequence substantiallyhomologous thereto; and(c) a light chain CDR3 domain comprising the amino acid sequence of SEQID NO: 6, 12, 18, 24, 112, 133, 134, 137 or 138 or a sequencesubstantially homologous thereto.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6 or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 1 or a sequence substantially homologous thereto and/or SEQ IDNO: 4 or a sequence substantially homologous thereto, and/or a CDR2domain comprising the amino acid sequence of SEQ ID NO: 2 or a sequencesubstantially homologous thereto and/or SEQ ID NO: 5 or a sequencesubstantially homologous thereto, are also present.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:12 or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 7 or a sequence substantially homologous thereto and/or SEQ IDNO: 10 or a sequence substantially homologous thereto, and/or SEQ ID NO:101 or a sequence substantially homologous thereto, and/or a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 8 or a sequencesubstantially homologous thereto and/or SEQ ID NO: 11 or a sequencesubstantially homologous thereto, are also present.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:15 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:18 or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 1 or a sequence substantially homologous thereto and/or SEQ IDNO: 16 or a sequence substantially homologous thereto, and/or a CDR2domain comprising the amino acid sequence of SEQ ID NO: 2 or a sequencesubstantially homologous thereto and/or SEQ ID NO: 17 or a sequencesubstantially homologous thereto, are also present.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:21 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 19 or a sequence substantially homologous thereto and/or SEQ IDNO: 22 or a sequence substantially homologous thereto, and/or a CDR2domain comprising the amino acid sequence of SEQ ID NO: 20 or a sequencesubstantially homologous thereto and/or SEQ ID NO: 23 or a sequencesubstantially homologous thereto, are also present.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:112 or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 7 or a sequence substantially homologous thereto and/or SEQ IDNO: 108 or a sequence substantially homologous thereto, and/or a CDR2domain comprising the amino acid sequence of SEQ ID NO: 8 or a sequencesubstantially homologous thereto and/or SEQ ID NO: 110 or a sequencesubstantially homologous thereto, are also present.

In certain preferred embodiments, the antibody that binds to binds toCCR4 and which is capable of inhibiting the binding of MDC to CCR4comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:133, 134, 137 or 138, or a sequence substantially homologous thereto.

More preferably, a CDR1 domain comprising the amino acid sequence of SEQID NO: 125 or 126 or a sequence substantially homologous thereto and/orSEQ ID NO: 129 or 130 or a sequence substantially homologous thereto,and/or a CDR2 domain comprising the amino acid sequence of SEQ ID NO:127 or 128 or a sequence substantially homologous thereto and/or SEQ IDNO: 131 or 132 or a sequence substantially homologous thereto, are alsopresent.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 1 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, or a sequence substantially homologousthereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 4 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 5 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 6 or a sequencesubstantially homologous thereto, are present individually or incombination.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 7 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, or a sequence substantially homologousthereto, are present individually or in combination.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 101 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 9, or a sequence substantially homologousthereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 10 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 11 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 12 or a sequencesubstantially homologous thereto, are present individually or incombination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 108 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 110 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 112 or a sequencesubstantially homologous thereto, are present individually or incombination.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 1 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 15, or a sequence substantially homologousthereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 16 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 17 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 18 or a sequencesubstantially homologous thereto, are present individually or incombination.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 19 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 20 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 21, or a sequence substantially homologousthereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 22 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 23 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 24 or a sequencesubstantially homologous thereto, are present individually or incombination.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 125 or 126 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 127 or 128 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 9, or a sequencesubstantially homologous thereto, are present individually or incombination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 129 or 130 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO: 131 or 132 or a sequence substantially homologous thereto, and CDR3comprising the amino acid sequence of SEQ ID NO: 133, 134, 137 or 138 ora sequence substantially homologous thereto, are present individually orin combination.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to CCR4 and which is capable ofinhibiting the binding of MDC to CCR4 comprising a heavy chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 6 or a sequencesubstantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 2 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 1 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 4 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 9 or a sequence substantially homologous theretoand/or a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 12 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 8 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 11 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 10 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 9 or a sequence substantially homologous theretoand/or a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 112 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 8 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 110 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 or 101 ora sequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 108 or asequence substantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 15 or a sequence substantially homologous theretoand/or a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 18 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 2 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 17 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 1 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 16 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 21 or a sequence substantially homologous theretoand/or a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 24 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 20 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 23 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 19 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 22 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 9 or a sequence substantially homologous theretoand/or a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 133, 134, 137 or 138 or a sequence substantially homologousthereto.

Said antibody optionally further comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 127 or 128 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 131 or 132 or asequence substantially homologous thereto and/or further comprises aheavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:125 or 126 or a sequence substantially homologous thereto and/or a lightchain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 129or 130 or a sequence substantially homologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to binds to CCR4 and which is capable ofinhibiting the binding of MDC to CCR4 comprising a heavy chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 2 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5 or a sequencesubstantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 6 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 1 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 4 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR2 domain comprising the amino acidsequence of SEQ ID NO: 8, 127 or 128 or a sequence substantiallyhomologous thereto and/or a light chain CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 11, 131 or 132 or a sequence substantiallyhomologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 9 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, 133, 134, 137 or138 or a sequence substantially homologous thereto and/or furthercomprises a heavy chain CDR1 domain comprising the amino acid sequenceof SEQ ID NO: 7, 101, 125 or 126 or a sequence substantially homologousthereto and/or a light chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 10, 129 or 130 or a sequence substantiallyhomologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to binds to CCR4 and which is capable of inhibiting the binding ofMDC to CCR4 comprising a heavy chain CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 8 or a sequence substantially homologousthereto and/or a light chain CDR2 domain comprising the amino acidsequence of SEQ ID NO: 110 or a sequence substantially homologousthereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 9 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 112 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 108 or asequence substantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to binds to CCR4 and which is capable of inhibiting the binding ofMDC to CCR4 comprising a heavy chain CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 2 or a sequence substantially homologousthereto and/or a light chain CDR2 domain comprising the amino acidsequence of SEQ ID NO: 17 or a sequence substantially homologousthereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 15 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 18 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 1 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 16 or a sequencesubstantially homologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to binds to CCR4 and which is capable ofinhibiting the binding of MDC to CCR4 comprising a heavy chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 20 or a sequencesubstantially homologous thereto and/or a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 23 or a sequencesubstantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 21 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 24 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 19 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 22 or a sequencesubstantially homologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to CCR4 and which is capable ofinhibiting the binding of MDC to CCR4 comprising a heavy chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 1 or a sequencesubstantially homologous thereto and/or a light chain CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 4 or a sequencesubstantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 6 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR2 domain comprising the amino acid sequence of SEQ ID NO: 2 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7, 125 or 126 or a sequence substantiallyhomologous thereto and/or a light chain CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 10, 129 or 130 or a sequence substantiallyhomologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 9 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, 133, 134, 137 or138 or a sequence substantially homologous thereto and/or furthercomprises a heavy chain CDR2 domain comprising the amino acid sequenceof SEQ ID NO: 8, 127 or 128 or a sequence substantially homologousthereto and/or a light chain CDR2 domain comprising the amino acidsequence of SEQ ID NO: 11, 131 or 132 or a sequence substantiallyhomologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to CCR4 and which is capable ofinhibiting the binding of MDC to CCR4 comprising a heavy chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 101 or asequence substantially homologous thereto and/or a light chain CDR1domain comprising the amino acid sequence of SEQ ID NO: 10 or a sequencesubstantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 9 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR2 domain comprising the amino acid sequence of SEQ ID NO: 8 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 11 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7 or a sequence substantially homologous theretoand/or a light chain CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 108 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 9 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 112 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR2 domain comprising the amino acid sequence of SEQ ID NO: 8 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 110 or asequence substantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 1 or a sequence substantially homologous theretoand/or a light chain CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 16 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 15 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 18 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR2 domain comprising the amino acid sequence of SEQ ID NO: 2 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 17 or a sequencesubstantially homologous thereto.

In certain embodiments, the present invention provides an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 19 or a sequence substantially homologous theretoand/or a light chain CDR1 domain comprising the amino acid sequence ofSEQ ID NO: 22 or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 21 or a sequencesubstantially homologous thereto and/or a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 24 or a sequencesubstantially homologous thereto and/or further comprises a heavy chainCDR2 domain comprising the amino acid sequence of SEQ ID NO: 20 or asequence substantially homologous thereto and/or a light chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 23 or a sequencesubstantially homologous thereto.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5and 6 or a sequence substantially homologous to any one of the foregoingSEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10,11 and 12 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 101, 8, 9,10, 11 and 12 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 7, 8, 9, 108,110 and 112 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 1, 2, 15, 16,17 and 18 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 19, 20, 21,22, 23 or 24 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 125, 127, 9,129, 131 or 133 (137) or a sequence substantially homologous to any oneof the foregoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs: 126, 128, 9,130, 132 or 134 (138) or a sequence substantially homologous to any oneof the foregoing SEQ ID NOs.

Certain preferred antibodies comprise two or more of the light chainCDRs of SEQ ID NOs: 4, 5 and 6, or 10, 11 and 12, or 16, 17 and 18, or22, 23 and 24, or 108, 110 and 112, or 129, 131 and 133 (137), or 130,132 and 134 (138); or sequences substantially homologous to any one ofthe foregoing SEQ ID NOs.

Especially preferred binding molecules comprise 3 of the light chainCDRs of SEQ ID NOs: 4, 5 and 6; or 10, 11 and 12; or 16, 17 and 18; or22, 23 and 24; or 108, 110 and 112, or 129, 131 and 133 (137), or 130,132 and 134 (138); or sequences substantially homologous to any one ofthe foregoing SEQ ID NOs (i.e. one of each of the aforementioned lightchain CDR1 and CDR2 and CDR3 or sequences substantially homologousthereto).

Other certain preferred antibodies comprise two or more of the heavychain CDRs of SEQ ID NOs: 1, 2 and 3; or 7, 8 and 9; or 1, 2 and 15; or19, 20 and 21; or 101, 8 and 9, or 125 and 127 and 9, or 126 and 128 and9; or sequences substantially homologous to any one of the foregoing SEQID NOs.

Especially preferred antibodies comprise 3 of the heavy chain CDRs ofSEQ ID NOs: 1, 2 and 3; or 7, 8 and 9; or 1, 2 and 15; or 19, 20 and 21;or 101, 8 and 9, or 125 and 127 and 9, or 126 and 128 and 9; orsequences substantially homologous to any one of the foregoing SEQ IDNOs (i.e. one of each of the aforementioned heavy chain CDR1 and CDR2and CDR3 or sequences substantially homologous thereto).

In some embodiments, the combination of a heavy chain CDR1 of SEQ ID NO:1 with a heavy chain CDR2 of SEQ ID NO: 2 is particularly preferred.

In some embodiments, the combination of a heavy chain CDR3 of SEQ ID NO:9 with a heavy chain CDR2 of SEQ ID NO: 8 and/or a heavy chain CDR1 ofSEQ ID NO: 7 or 101 is particularly preferred.

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 4, 5 and 6 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 1, 2 and 3, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 10, 11 and 12 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 7, 8 and 9, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 10, 11 and 12 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 101, 8 and 9, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 108, 110 and 112 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 7, 8 and 9, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 16, 17 and 18 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 1, 2 and 15, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 22, 23 and 24 or sequences substantiallyhomologous to any one of these sequences (i.e. one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 19, 20 and 21, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs: 129, 131 and 133 (137) or sequencessubstantially homologous to any one of these sequences (i.e. one of eachof the aforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs: 125, 127 and 9, or sequences substantially homologous any one ofthese sequences (i.e. one of each of the aforementioned heavy chain CDR1and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 1, a heavy chain CDR2 domain of SEQ ID NO: 2, and aheavy chain CDR3 domain of SEQ ID NO: 3, or sequences substantiallyhomologous to any one of the aforementioned sequences; and/or comprise

a light chain CDR1 domain of SEQ ID NO: 4, a light chain CDR2 domain ofSEQ ID NO: 5, and a light chain CDR 3 domain of SEQ ID NO: 6, orsequences substantially homologous to any one of the aforementionedsequences.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 7 or 101, a heavy chain CDR2 domain of SEQ ID NO:8, and a heavy chain CDR3 domain of SEQ ID NO: 9, or sequencessubstantially homologous to any one of the aforementioned sequences;and/or comprise

a light chain CDR1 domain of SEQ ID NO: 10, a light chain CDR2 domain ofSEQ ID NO: 11, and a light chain CDR 3 domain of SEQ ID NO: 12, orsequences substantially homologous to any one of the aforementionedsequences.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 7, a heavy chain CDR2 domain of SEQ ID NO: 8, and aheavy chain CDR3 domain of SEQ ID NO: 9, or sequences substantiallyhomologous to any one of the aforementioned sequences; and/or comprise alight chain CDR1 domain of SEQ ID NO: 108, a light chain CDR2 domain ofSEQ ID NO: 110, and a light chain CDR 3 domain of SEQ ID NO: 112, orsequences substantially homologous to any one of the aforementionedsequences.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 1, a heavy chain CDR2 domain of SEQ ID NO: 2, and aheavy chain CDR3 domain of SEQ ID NO: 15, or sequences substantiallyhomologous to any one of the aforementioned sequences; and/or comprise alight chain CDR1 domain of SEQ ID NO: 16, a light chain CDR2 domain ofSEQ ID NO: 17, and a light chain CDR 3 domain of SEQ ID NO: 18, orsequences substantially homologous to any one of the aforementionedsequences.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 19, a heavy chain CDR2 domain of SEQ ID NO: 20, anda heavy chain CDR3 domain of SEQ ID NO: 21, or sequences substantiallyhomologous to any one of the aforementioned sequences; and/or comprise alight chain CDR1 domain of SEQ ID NO: 22, a light chain CDR2 domain ofSEQ ID NO: 23, and a light chain CDR 3 domain of SEQ ID NO: 24, orsequences substantially homologous to any one of the aforementionedsequences.

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO: 125, 126, 135 or 136, a heavy chain CDR2 domain ofSEQ ID NO: 127 or 128, and a heavy chain CDR3 domain of SEQ ID NO: 9, orsequences substantially homologous to any one of the aforementionedsequences; and/or comprise a light chain CDR1 domain of SEQ ID NO: 129or 130, a light chain CDR2 domain of SEQ ID NO: 131 or 132, and a lightchain CDR 3 domain of SEQ ID NO: 133 (137) or 134 (138), or sequencessubstantially homologous to any one of the aforementioned sequences.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3 or 15.In a preferred aspect of this embodiment, one or more of said lightchain variable region CDRs are selected from the group consisting of:(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 4 or 16,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5 or 17,and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6 or 18.Preferably, 2 or 3 of said light chain variable region CDRs are selectedfrom the above group.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 7 or 101,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 8, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9.

In a preferred aspect of this embodiment, one or more of said lightchain variable region CDRs are selected from the group consisting of:

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 10,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 11, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 12.Preferably, 2 or 3 of said light chain variable region CDRs are selectedfrom the above group.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 7,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 8, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9.

In a preferred aspect of this embodiment, one or more of said lightchain variable region CDRs are selected from the group consisting of:

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 108,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 110, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 112.Preferably, 2 or 3 of said light chain variable region CDRs are selectedfrom the above group.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 19,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 20, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 21.

In a preferred aspect of this embodiment, one or more of said lightchain variable region CDRs are selected from the group consisting of:

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 22,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 23, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 24.Preferably, 2 or 3 of said light chain variable region CDRs are selectedfrom the above group.

In a further embodiment, the invention provides an antibody that bindsto CCR4 and which is capable of inhibiting the binding of MDC to CCR4and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said heavy chain variable regioncomprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 125, 126, 135 or 136,(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 127 or128, and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9.

In a preferred aspect of this embodiment, one or more of said lightchain variable region CDRs are selected from the group consisting of:

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 129 or 130,(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 131 or132, and(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 133 (137)or 134 (138). Preferably, 2 or 3 of said light chain variable regionCDRs are selected from the above group.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises:

a VH domain that comprises one, two or three of the heavy chain CDRs ofSEQ ID NOs: 1, 2 or 3, or sequences substantially homologous to one ormore of SEQ ID NOs: 1, 2 or 3, and/or a VL domain that comprises one,two or three of the light chain CDRs of SEQ ID NOs: 4, 5 or 6, orsequences substantially homologous to one or more of SEQ ID NOs: 4, 5 or6.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VH domain that comprisesone, two or three of the heavy chain CDRs of SEQ ID NOs: 7, 8, 9 or 101,or sequences substantially homologous to one or more of SEQ ID NOs: 7,8, 9 or 101, and/or a VL domain that comprises one, two or three of thelight chain CDRs of SEQ ID NOs: 10, 11 or 12, or sequences substantiallyhomologous to one or more of SEQ ID NOs: 10, 11 or 12.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VH domain that comprisesone, two or three of the heavy chain CDRs of SEQ ID NOs: 7, 8 or 9, orsequences substantially homologous to one or more of SEQ ID NOs: 7, 8 or9, and/or a VL domain that comprises one, two or three of the lightchain CDRs of SEQ ID NOs: 108, 110 or 112, or sequences substantiallyhomologous to one or more of SEQ ID NOs: 108, 110 or 112.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VH domain that comprisesone, two or three of the heavy chain CDRs of SEQ ID NOs: 1, 2 or 15, orsequences substantially homologous to one or more of SEQ ID NOs: 1, 2 or15, and/or a VL domain that comprises one, two or three of the lightchain CDRs of SEQ ID NOs: 16, 17 or 18, or sequences substantiallyhomologous to one or more of SEQ ID NOs: 16, 17 or 18.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VH domain that comprisesone, two or three of the heavy chain CDRs of SEQ ID NOs: 19, 20 or 21,or sequences substantially homologous to one or more of SEQ ID NOs: 19,20 or 21, and/or a VL domain that comprises one, two or three of thelight chain CDRs of SEQ ID NOs: 22, 23 or 24, or sequences substantiallyhomologous to one or more of SEQ ID NOs: 22, 23 or 24.

Certain further preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VH domain that comprisesone, two or three of the heavy chain CDRs of SEQ ID NOs: 125, 127 or 9,or sequences substantially homologous to one or more of SEQ ID NOs: 125,127 or 9, and/or a VL domain that comprises one, two or three of thelight chain CDRs of SEQ ID NOs: 129, 131 or 133 (137), or sequencessubstantially homologous to one or more of SEQ ID NOs: 129, 131 or(137).

More especially preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VL domain that comprises 3light chain CDRs and a VH domain that comprises 3 heavy chain CDRs ofSEQ ID NOs: 1, 2, and 3 or 15. In preferred embodiments one, two orthree of the light chain CDRs are as defined in SEQ ID NOs: 4, 5 and 6,or 16, 17 and 18.

More especially preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VL domain that comprises 3light chain CDRs and a VH domain that comprises 3 heavy chain CDRs ofSEQ ID NOs: 8, 9, and 7 or 101. In preferred embodiments one, two orthree of the light chain CDRs are as defined in SEQ ID NOs: 10, 11 and12, or 108, 110 and 112.

More especially preferred embodiments of the invention provide anantibody that binds to CCR4 and which is capable of inhibiting thebinding of MDC to CCR4 and that comprises: a VL domain that comprises 3light chain CDRs and a VH domain that comprises 3 heavy chain CDRs ofSEQ ID NOs: 125, 127 and 9. In preferred embodiments one, two or threeof the light chain CDRs are as defined in SEQ ID NOs: 129, 131 and 133(137).

In all the embodiments of the present invention as described herein, X,where present in an amino acid sequence, represents a variable aminoacid. Thus, where a sequence comprises more than one X, each X may bedifferent, but one or more X may also be the same. Preferably, atposition 1 of SEQ ID NO: 125, X=S or N; and/or at position 4 of SEQ IDNO: 125, X=I or M; and/or at position 1 of SEQ ID NO: 127, X=G or A;and/or at position 3 of SEQ ID NO: 127, X=I or S; and/or at position 5of SEQ ID NO: 127, X=I or S; and/or at position 6 of SEQ ID NO: 127, X=For G; and/or at position 8 of SEQ ID NO: 127, X=T or S; and/or atposition 9 of SEQ ID NO: 127, X=A or T and/or at position 3 of SEQ IDNO: 129, X=S or G; and/or at position 4 of SEQ ID NO: 129, X=T or G;and/or at position 9 of SEQ ID NO: 129, X=S or R; and/or at position 10of SEQ ID NO: 129, X=R or H; and/or at position 11 of SEQ ID NO: 129,X=Y or F; and/or at position 13 of SEQ ID NO: 129, X=Y or F; and/or atposition 3 of SEQ ID NO: 131, X=H or N; and/or at position 2 of SEQ IDNO: 133 or 137, X=A or V; and/or at position 6 of SEQ ID NO: 133 or 137,X=S or T.

A preferred embodiment of SEQ ID NO: 125 is SEQ ID NO: 126, a preferredembodiment of SEQ ID NO: 127 is SEQ ID NO: 128, a preferred embodimentof SEQ ID NO: 129 is SEQ ID NO: 130, a preferred embodiment of SEQ IDNO:131 is SEQ ID NO: 132, and a preferred embodiment of SEQ ID NO:133 isSEQ ID NO: 134.

A preferred embodiment of SEQ ID NO:125 and/or SEQ ID NO: 126 is SEQ IDNO: 135, more preferably SEQ ID NO:136. A preferred embodiment of SEQ IDNO: 133 is SEQ ID NO: 137 or SEQ ID NO:138. A preferred embodiment ofSEQ ID NO: 134 is SEQ ID NO:138.

Embodiments in which a heavy chain CDR1 of SEQ ID NO: 125 is present arepreferred. Also preferred are embodiments in which a heavy chain CDR1 ofSEQ ID NO: 101 is present. Thus, the heavy chain CDR1 preferably startswith the amino acid S (serine).

Certain preferred embodiments of the invention provide an antibody thatbinds to CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising a VH domain that has the amino acid sequence of SEQ IDNO: 139, 69, 71, 73, 75 or 105 or a sequence substantially homologousthereto and/or a VL domain that has the amino acid sequence of SEQ IDNO: 140, 70, 72, 74, 76 or 115 or a sequence substantially homologousthereto. Preferably, said VH and/or VL domains have at least 1, 2, 3, 4,or 5, e.g. 6 of the CDR sequences disclosed herein.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 139, 69, 71,73, 75 or 105 and a VL domain that comprises 3 light chain CDRs.Preferably said light chain CDRs have SEQ ID NOs 4, 5 and 6, or 10, 11and 12, or 16, 17 and 18, or 22, 23 and 24, or 108, 110 and 112, or 129,131 and 133 (137), or 130, 132 and 134 (138).

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 69 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 70 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 71 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 72 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 71 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 115 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 105 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 72 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 73 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 74 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 75 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 76 or a sequence substantiallyhomologous thereto.

Further preferred embodiments provide an antibody that binds to CCR4 andwhich is capable of inhibiting the binding of MDC to CCR4 comprising aVH domain that has the amino acid sequence of SEQ ID NO: 139 or asequence substantially homologous thereto and/or a VL domain that hasthe amino acid sequence of SEQ ID NO: 140 or a sequence substantiallyhomologous thereto.

In a yet further embodiment, the present invention provides an antibodythat binds CCR4 and which is capable of inhibiting the binding of MDC toCCR4 comprising the amino acid sequence of SEQ ID NO: 35 (said antibodyalso being referred to herein as 17G scFv), SEQ ID NO: 46 (said antibodyalso being referred to herein as 9E scFv), SEQ ID NO: 104 (said antibodyalso being referred to herein as 9E10J scFv), SEQ ID NO: 114 (saidantibody also being referred to herein as 9E1 D scFv), SEQ ID NO: 57(said antibody also being referred to herein as 1O scFv), or SEQ ID NO:68 (said antibody also being referred to herein as 11F scFv), orcomprising a fragment of any thereof that binds CCR4 and which iscapable of inhibiting the binding of MDC to CCR4, or a sequencesubstantially homologous to any of the above sequence.

The invention is exemplified by monoclonal antibodies 17G, 9E, 1O, 11F,9E10J and 9E1 D, single chain forms of which are shown in Tables 1, 2,3, 4, 11 and 12 (SEQ ID NOs: 35, 46, 57, 68, 103 and 114 respectively).Full length IgG forms of antibodies 17G, 9E, 1O and 11F have been madeand their sequences are shown in Tables 5, 6, 7 and 8 respectively. TheCDR domains, VH and VL domains of the 17G, 9E, 11F and 1O antibodies areshown in Tables 1 to 4 and FIGS. 1-4 and CDR domains, VH and VL domainsof the 9E10J and 9E1 D antibodies are shown in Tables 11 and 12. Fulllength IgG forms of antibodies 9E10J and 9E1 D have been made and theirsequences are shown in Tables 13 and 14. Table 23 lists consensussequences. Antibodies comprising these CDR domains or VH and VL domains(or sequences substantially homologous thereto) are preferred aspects ofthe invention.

A preferred embodiment of the invention is a scFv form of the 17Gantibody comprising or consisting of SEQ ID NO: 35, which is preferablyencoded by SEQ ID NO: 34. More preferably, the antibody comprises orconsists of the amino acid sequence shown in FIG. 1 and preferably thisantibody is encoded by the nucleic acid sequence shown in FIG. 1.

Another preferred embodiment of the invention is a scFv form of the 9Eantibody comprising or consisting of SEQ ID NO: 46, which is preferablyencoded by SEQ ID NO: 45. More preferably, the antibody comprises orconsists of the amino acid sequence shown in FIG. 2 and preferably thisantibody is encoded by the nucleic acid sequence shown in FIG. 2.

Another preferred embodiment of the invention is a scFv form of the9E10J antibody comprising or consisting of SEQ ID NO: 104, which ispreferably encoded by SEQ ID NO: 103. More preferably, the antibodycomprises or consists of the amino acid sequence shown in FIG. 27 andpreferably this antibody is encoded by the nucleic acid sequence shownin FIG. 27.

Another preferred embodiment of the invention is a scFv form of the 9E1D antibody comprising or consisting of SEQ ID NO: 114, which ispreferably encoded by SEQ ID NO: 113. More preferably, the antibodycomprises or consists of the amino acid sequence shown in FIG. 28 andpreferably this antibody is encoded by the nucleic acid sequence shownin FIG. 28.

Another preferred embodiment of the invention is a scFv form of the 1Oantibody comprising or consisting of SEQ ID NO: 57, which is preferablyencoded by SEQ ID NO: 56. More preferably, the antibody comprises orconsists of the amino acid sequence shown in FIG. 3 and preferably thisantibody is encoded by the nucleic acid sequence shown in FIG. 3.

Another preferred embodiment of the invention is a scFv form of the 11Fantibody comprising or consisting of SEQ ID NO: 68, which is preferablyencoded by SEQ ID NO: 67. More preferably, the antibody comprises orconsists of the amino acid sequence shown in FIG. 4 and preferably thisantibody is encoded by the nucleic acid sequence shown in FIG. 4.

Other preferred embodiments are IgG forms of the 17G, 9E, 1O, 11F, 9EJ10and 9E1 D antibodies, preferably full length IgG forms. The IgG1 form ofany of these antibodies is most preferred.

Thus, another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 87 (amino acid)and/or a light chain of SEQ ID NO: 88 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO:85and/or a light chain encoded by SEQ ID NO:86. It is of course understoodthat full IgG antibodies will comprise two substantially identical heavychains and two substantially identical light chains.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 91 (amino acid)and/or a light chain of SEQ ID NO: 92 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 89and/or a light chain encoded by SEQ ID NO: 90.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 95 (amino acid)and/or a light chain of SEQ ID NO: 96 (amino acid). Also preferred is anIgG antibody which comprises a heavy chain encoded by SEQ ID NO: 93and/or a light chain encoded by SEQ ID NO: 94.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 99 (amino acid)and/or a light chain of SEQ ID NO: 100 (amino acid). Also preferred isan IgG antibody which comprises a heavy chain encoded by SEQ ID NO: 97and/or a light chain encoded by SEQ ID NO: 98.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 119 (amino acid)and/or a light chain of SEQ ID NO: 120 (amino acid). Also preferred isan IgG antibody which comprises a heavy chain encoded by SEQ ID NO: 117and/or a light chain encoded by SEQ ID NO: 118.

Another preferred embodiment of the invention is a full length IgGantibody which comprises a heavy chain of SEQ ID NO: 123 (amino acid)and/or a light chain of SEQ ID NO: 124 (amino acid). Also preferred isan IgG antibody which comprises a heavy chain encoded by SEQ ID NO: 121and/or a light chain encoded by SEQ ID NO: 122.

It is believed that the antibodies of the invention may bind to adifferent epitope to the known anti-CCR4 antibody family which comprisesKM2160, KM3060, KM2760 and KW-0761. Antibody KM2160 is a murine antibodywhich was raised against a peptide fragment of CCR4 and which recognisesan epitope existing in a region of positions 2-29 from the N-terminalamino acid of human CCR4 (EP1270595). KM2760 is a chimeric version ofthe antibody having the same binding characteristics (EP1270595).Antibody KM3060 is identical to KM2760, except that it is highlyfucosylated (Niwa et al. 2004, Cancer Research 64, 2127-2133). KW-0761is a humanised version of KM2760 (Ishida et al. Annals of Oncology 2008,vol 19, supplement 4, 513).

KM2760 has been reported not to block the interaction between CCR4 andTARC or MDC (Ishida et al. 2006, Cancer Research 66 (11), pp 5716-5722),which is consistent with the inventors' findings using the equivalentantibody KM3060var (corresponding to KM3060, but potentially having adifferent sugar profile as it was expressed in a different host) shownin Example 3. By contrast, the antibodies of the present invention werefound to block the interaction between CCR4 and MDC and the interactionbetween CCR4 and TARC (see Example 3). This strongly suggests that theantibodies of the invention bind to a different epitope than the priorart family which comprises KM2160, KM3060, KM2760 and KW-0761.

Moreover, antibodies 17G and 9E were found to compete with one anotherfor binding to CCR4, indicating that they bind to the same, similar orat least overlapping epitopes. Neither of these antibodies competes withKM3060var for binding to CCR4, indicating that KM3060var binds to adifferent epitope.

It is also believed that the antibodies of the invention may bind to adifferent epitope to the commercially available anti-CCR4 antibody 1 G1.BD Pharmingen make it clear on the technical data sheet for thisantibody that this antibody is not a neutralising antibody. By contrast,the antibodies of the present invention are capable of blocking thebinding of MDC and TARC to CCR4 and inhibiting the MDC or TARC-inducedincrease in intracellular calcium ions. This strongly suggests that theantibodies of the invention bind to a different epitope than the 1 G1antibody.

Thus, also provided are antibodies which can compete with any of theantibodies described herein for binding to CCR4.

The term “competing antibodies”, as used herein, refers to antibodiesthat bind to about, substantially or essentially the same, or even thesame, epitope as a “reference antibody”. “Competing antibodies” includeantibodies with overlapping epitope specificities. Competing antibodiesare thus able to effectively compete with a reference antibody forbinding to CCR4. Preferably, the competing antibody can bind to the sameepitope as the reference antibody. Alternatively viewed, the competingantibody preferably has the same epitope specificity as the referenceantibody.

“Reference antibodies” as used herein are antibodies which can bind toan epitope in the extracellular domain of human CCR4 and which have oneor more of the CDR sequences are defined herein, preferably a VH and aVL domain as defined herein, more preferably a VH of SEQ ID NO: 130 anda VL of SEQ ID NO: 140, or a VH of SEQ ID NO: 69 and a VL of SEQ ID NO:70, or a VH of SEQ ID NO: 71 and a VL of SEQ ID NO: 72, or a VH of SEQID NO: 105 and a VL of SEQ ID NO: 72, or a VH of SEQ ID NO: 71 and a VLof SEQ ID NO: 115, or a VH of SEQ ID NO: 73 and a VL of SEQ ID NO: 74,or a VH of SEQ ID NO: 75 and a VL of SEQ ID NO: 76. Most preferredreference antibodies are selected from 17G, 9E, 11F, 1O, 9E10J and 9E1D.

The identification of one or more competing antibodies is astraightforward technical matter now that reference antibodies such as17G, 9E, 11F, 1O, 9E10J and 9E1 D have been provided. As theidentification of competing antibodies is determined in comparison to areference antibody, it will be understood that actually determining theepitope to which either or both antibodies bind is not in any wayrequired in order to identify a competing antibody. However, epitopemapping can be performed using standard techniques, if desired.

By way of example, the following methods for the identification anddefinition of epitopes are mentioned herein. The amino acid sequence ofCCR4 is known, so synthetic peptides may be used for epitope mapping,e.g. using the Pepscan assay. Site directed mutagenesis is also apowerful tool in epitope mapping and can be used to evaluate the role ofsingle amino acids in immune complex formation. Protein footprintingrelies on the fact that the epitope is protected from cleavage whenbound as an antibody-antigen complex. Enzyme linked immunosorbent assay(ELISA) and haemaglutination and slot-blotting may also be used inepitope mapping. Crystallisation of the antigen with the antibody may beused to map a non-linear epitope. Protocols for carrying out suchmethods are widely available and the skilled person will be aware ofsuitable alternative methods of epitope mapping.

The identification of competing antibodies can be readily determinedusing any one of variety of immunological screening assays in whichantibody competition can be assessed. All such assays are routine in theart and are further described herein in detail. Each of U.S. Pat. Nos.6,342,219, 6,524,583, 7,056,509, 6,887,468, 6,342,221, 6,676,941,6,703,020 and 6,416,758 are specifically incorporated herein byreference for purposes including even further supplementing the presentteaching concerning how to identify competing antibodies. Example 4 ofthe present specification discloses a suitable competition assay.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different isotype, a simplecompetition assay may be employed in which the reference and testantibodies are admixed (or pre-adsorbed) and applied to CCR4-containingcomposition, preferably cells expressing CCR4, phage displaying CCR4, orbiochips containing immobilised CCR4. Protocols based upon ELISAs areparticularly suitable for use in such simple competition studies.

In certain embodiments, one would pre-mix the reference antibodies(e.g., 17G, 9E, 11F, 1O, 9E10J and 9E1 D) with varying amounts of thetest antibodies (e.g., 1:10, 1:100 or 1:1000) for a period of time priorto applying to an antigen composition. In other embodiments, thereference and varying amounts of test antibodies can simply be admixedduring exposure to the antigen composition. In any event, by usingspecies or isotype secondary antibodies one will be able to detect onlythe bound reference antibodies, the binding of which will be reduced bythe presence of a test antibody that “competes” for binding.

In conducting an antibody competition study between a reference antibodyand any test antibody (irrespective of species or isotype), one mayfirst label the reference (e.g., 17G, 9E, 11F, 1O, 9E10J and 9E1 D) witha detectable label, such as, e.g., biotin or an enzymatic or radioactivelabel to enable subsequent identification. In these cases, one wouldpre-mix or incubate the labeled reference antibodies with the testantibodies to be examined at various ratios (e.g., 1:10, 1:100 or1:1000) and (optionally after a suitable period of time) then assay thereactivity of the labeled reference antibodies and compare this with acontrol value in which no potentially competing test antibody wasincluded in the incubation.

The assay may be any one of a range of immunological assays based uponantibody binding, and the reference antibodies would be detected bymeans of detecting their label, e.g., using streptavidin in the case ofbiotinylated antibodies or by using a chromogenic substrate inconnection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label. An antibody that competes withthe reference antibodies for binding to CCR4 will be able to effectivelyor significantly reduce reference antibody binding to CCR4, as evidencedby a reduction in bound label.

The reactivity of the (labeled) reference antibodies in the absence of acompletely irrelevant antibody would be the control high value. Thecontrol low value would be obtained by incubating the labeled reference(e.g., 17G, 9E, 11F, 1O, 9E10J or 9E1 D) antibodies with unlabelledantibodies of exactly the same type, when competition would occur andreduce binding of the labeled antibodies. In a test assay, a significantreduction in labeled antibody reactivity in the presence of a testantibody is indicative of a test antibody that “competes” with thelabeled antibody for binding to CCR4.

A significant reduction is a “reproducible”, i.e., consistentlyobserved, reduction in binding. A “significant reduction” in terms ofthe present application is defined as a reproducible reduction (inbinding of the reference antibody to CCR4 in an ELISA) of at least about20%, more preferably at least about 25, 30, 35, 40, 45, 50, 55, 60 or65%, even more preferably at least about 70%, about 75% or about 80% atany ratio between about 1:10 and about 1:100. Antibodies with even morestringent competing activities will exhibit a reproducible reduction (inbinding of the reference antibody to CCR4 in an ELISA or other suitableassay) of at least about 82%, about 85%, about 88%, about 90%, about 92%or about 95% or so at any ratio between about 1:10 and about 1:100.Complete or near-complete competition, such as exhibiting a reproduciblereduction in binding of the reference antibody to CCR4 of about 99%,about 98%, about 97% or about 96% or so, although by no means requiredto practice the invention, is certainly not excluded.

The method described above is only one example of a suitable competitionassay. The skilled person will be aware of other suitable methods andvariations. An alternative competition assay is described below.

Before the alternative competition assay is performed using flowcytometry, some quantities of the tested antibody should be labeled,e.g. by biotinylation. The functionality (retention of the cell-bindingproperties) of the biotinylated product and the minimal concentration ofthe biotinylated antibody of the invention (Ab1) that gives sub-maximalbinding against a fixed number of CCR4+ cells is determined. A total of10⁶ cells are harvested from exponentially growing cultures andincubated with various antibody concentrations for a suitable period oftime at a suitable temperature, e.g. 1 hr at 4° C. The cells are washedand incubated with a suitable detection antibody for a suitable periodof time at a suitable temperature, e.g. an additional hour at 4° C.After washing, the cells are analyzed by flow cytometry. For each testantibody, a saturation curve is generated from the data by plottingmedian fluorescence intensity (MFI) against the antibody concentration.

For the alternative competition assay, CCR4+ cells may be prepared asabove and treated in duplicate with a mixture of fixed concentration oflabeled (biotinylated) antibody (bio-Ab1) and increasing concentrationsof non-labeled competitive antibody. The fixed concentration is theminimal concentration of antibody that generates reasonable fluorescencesignal against a fixed number of tumor cells as determined above.Ideally, this fixed concentration in nM should be below the affinity ofthe treated antibody at equilibrium (K_(D)). In this case the describedmethod can be used for estimation of affinities of competitiveantibodies (Schodin and Kranz, 1993, J Biol Chem 268:25755-7). Theantibody mixture is incubated with target cells for a suitable period oftime at a suitable temperature, e.g. 1 hr at 4²C. The cells are washedand the cell binding of biotinylated antibody is revealed by incubationwith FITC-labeled streptavidin. After subtracting the backgroundfluorescence (PBS-5% FCS) from the median fluorescence reading for eachtest sample (bio-Ab1+Ab2), the percent of inhibition is calculated foreach Ab2 concentration “c” according to the formula:

% inhibition=(1−MFI ^(bio-Ab1+Ab2″c″) /MFI ^(bio-Ab1))×100

is calculated.

Any antibodies which can bind to CCR4 and which are capable ofinhibiting the binding of MDC to CCR4 and which can compete with any ofthe antibodies described herein are contemplated, but preferredantibodies are set out below. Accordingly, in some preferred embodimentsthere is provided the following.

An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody

(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 4;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5; and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6; and/orwherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2; and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody

(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 10;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 11; and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 12;and/or wherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 7 or 101;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 8; and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9; or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody

(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 108;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 110;and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 112;and/or wherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 7;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 8; and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9; orb) is an antibody which can compete with antibody (a) for binding toCCR4.

An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody

(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 16;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 17; and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 18;and/or wherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 1;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2; and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 15; orb) is an antibody which can compete with antibody (a) for binding toCCR4.

An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody

(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 22;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 23; and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 24;and/or wherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 19;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 20; and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 21; or(b) is an antibody which can compete with antibody (a) for binding toCCR4.An antibody which binds to an epitope in the extracellular domain ofhuman CC chemokine receptor 4 (CCR4) and which is capable of inhibitingthe binding of MDC to CCR4, wherein said antibody(a) comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said light chain variable region comprises:(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO: 129 or 130;(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 131 or132; and/or(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 133, 134,137 or 138; and/orwherein said heavy chain variable region comprises(iv) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQID NO: 125, 126, 135 or 136;(v) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 127 or 128;and/or(vi) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9; or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 69 and a VL domain of SEQ ID NO: 70;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 71 and a VL domain of SEQ ID NO: 72;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 105 and a VL domain of SEQ ID NO: 72;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 71 and a VL domain of SEQ ID NO: 115;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 73 and a VL domain of SEQ ID NO: 74;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 75 and a VL domain of SEQ ID NO: 76;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

In one embodiment, the antibody

(a) has a VH domain of SEQ ID NO: 139 and a VL domain of SEQ ID NO: 140;or(b) is an antibody which can compete with antibody (a) for binding toCCR4.

Preferably, antibody (b) has one or more of the CDR sequences, VHdomains and/or VL domains described herein.

Preferably, antibody (b) can bind to the same epitope as antibody (a).

Certain examples of substantially homologous sequences are sequencesthat have at least 70% identity to the amino acid sequences disclosed.In certain embodiments, the antibodies of the invention that bind toCCR4 and which are capable of inhibiting the binding of MDC to CCR4comprise at least one light chain variable region that includes an aminoacid sequence region of at least about 75%, more preferably at leastabout 80%, more preferably at least about 85%, more preferably at leastabout 90% or 95% and most preferably at least about 97% amino acidsequence identity to the amino acid sequence of SEQ ID NO: 140, 70, 72,74, 76 or 115; and/or at least one heavy chain variable region thatincludes an amino acid sequence region of at least about 75%, morepreferably at least about 80%, more preferably at least about 85%, morepreferably at least about 90% or 95% and most preferably at least about97% amino acid sequence identity to the amino acid sequence of SEQ IDNO: 139, 69, 71, 73, 75 or 105.

Other preferred examples of substantially homologous sequences aresequences containing conservative amino acid substitutions of the aminoacid sequences disclosed.

Other preferred examples of substantially homologous sequences aresequences containing up to 1, 2, 3 or 4 preferably up to 1 or 2, alteredamino acids in one or more of the CDR regions disclosed. Suchalterations might be conservative or non-conservative amino acidsubstitutions, or a mixture thereof.

In all such embodiments, preferred alterations are conservative aminoacid substitutions.

In all embodiments, the antibodies containing substantially homologoussequences retain the ability to bind CCR4 and the ability to inhibit thebinding of MDC to CCR4.

Other embodiments of the present invention provide binding proteins thatbind to CCR4 and have the ability to inhibit the binding of MDC to CCR4and that comprise an antibody of the invention, a VH or VL domain of theinvention, or one or more of the CDRs of the invention. In a preferredembodiment, such binding proteins are antibodies.

Preferred antibodies of the invention comprise at least one heavy chainvariable region that comprises three CDRs and at least one light chainvariable region that comprises three CDRs. Exemplary and preferredsequences for these CDRs are described herein.

As used herein, the succinct term “CCR4”, unless otherwise specificallystated or made clear from the scientific terminology, means CC chemokinereceptor 4 (also known as CD194).

CCR4 may be free CCR4, e.g. recombinant or purified CCR4, but preferablyit is present in a native form, e.g. on the surface of a cell.

The antibodies or binding proteins of the invention can also bind tofragments of CCR4, in particular fragments comprising or consisting ofthe extracellular domain, or can bind to entities comprising CCR4 orfragments of CCR4. Indeed, the epitope of the antibodies of theinvention is located in the extracellular domain of CCR4.

“CCR4” may also refer to any form of CCR4, particularly as CCR4 isconserved across mammalian species. The antibodies or antibody fragmentsof the invention may thus bind to human, monkey (e.g. cynomolgusmonkey), cow (bovine), mouse, rat, hamster, ferret, guinea pig and/orrabbit CCR4, for example. Preferably, the antibodies or antibodyfragments of the invention will bind at least to human CCR4. Thus,unless stated otherwise, any reference herein to “CCR4” may be read tomean “human CCR4”. In certain preferred embodiments, the antibodies orantibody fragments of the invention will bind at least to human andmonkey (e.g. cynomologus monkey) CCR4. In other preferred embodimentsthe antibodies or antibody fragments of the invention will bind at leastto human and mouse CCR4. In other preferred embodiments the antibodiesor antibody fragments of the invention will bind at least to human,monkey and mouse CCR4. In other preferred embodiments the antibodies orantibody fragments of the invention will bind at least to human, monkey,guinea pig and mouse CCR4. Antibodies 9E and 9E10J can bind to human andmonkey CCR4, but not to murine CCR4 (Example 8), so in some preferredembodiments, the antibodies or antibody fragments of the invention willbind at least to human and monkey CCR4, but not to murine CCR4. In otherpreferred embodiments, the antibodies or antibody fragments of theinvention will bind to human and monkey CCR4, but not to murine CCR4,e.g. only to human and monkey CCR4.

As used herein, the term “that binds to CCR4” or “anti-CCR4” in thecontext of antibodies or antibody fragments of the present invention,means antibodies or antibody fragments that are capable of one or moreof the following; preferably, of more than one of the following; andmost preferably, of all of the following:

(a) bind to CCR4 expressed on the surface of a cell, e.g. as assessed byflow cytometry or immunohistochemistry;(b) bind to a conformationally dependent (e.g. non linear) CCR4 epitope,e.g. as assessed by binding to CCR4 in a Western blot under non-reducingconditions;(c) bind to free CCR4; e.g. recombinantly expressed CCR4, on a solidsupport, e.g. as assessed by ELISA assay or BIAcore assay;(d) bind at least to human CCR4, more preferably to human and monkeyCCR4 or to human and mouse CCR4, most preferably to human, monkey andmouse CCR4 or to human and monkey CCR4 but not mouse CCR4;(e) bind to human CCR4 with a binding affinity (Kd) of 10 nM or less,preferably 5 nM or less, more preferably 3 nM or less or 2 nM or less,most preferably 1 nM or less as also discussed elsewhere herein;(f) bind to human and monkey CCR4 or to human and mouse CCR4, preferablyto human and monkey CCR4 but not mouse CCR4, with similar affinities,e.g. with a Kd of 10 nM or less, preferably 5 nM or less, morepreferably 3 nM or less or 2 nM or less, for example 1 nM or less asalso discussed elsewhere herein;

Preferred antibodies or antibody fragments of the present invention arealso capable of one or more of the following; preferably, of more thanone of the following; and most preferably, of all of the followingfunctional properties:

(g) induce ADCC of CCR4+ cells as described elsewhere herein;(h) inhibit the binding of CCR4 to at least MDC and/or TARC, preferablyMDC and TARC, or preferably at least MDC and/or TARC and one or moreselected from RANTES, MCP-1 and MIP-1alpha(i) induce anti tumour effects in vivo;(j) localize to tumours upon administration to an animal with a tumour;(k) induce CDC of CCR4+ cells;(l) inhibit CCR4-mediated cellular responses to a CCR4 ligand,preferably inhibit the increase in intracellular calcium ionconcentration in response to a CCR4 ligand;(m) inhibit chemotaxis of CCR4+ cells towards a CCR4 ligand such as MDC

In the context of binding to CCR4+ cells, it should be understood thatthe antibodies of the present invention bind to CCR4+ cells and do notsignificantly bind to CCR4⁻ cells (as shown in Example 2).

The term “do not significantly bind to CCR4⁻ cells” should be understoodsuch that any binding of the antibody to CCR4⁻ cells does not prohibitthe use of said antibody for therapeutic or diagnostic purposes. Thus,by “insignificant” binding to CCR4⁻ cells is meant that the binding ofthe antibody to CCR4⁻ cells is weaker than its binding to one or moreCCR4⁺ cells. Some cross-reaction with normal cells may thus occur, butthis level of binding can be considered to be “background” binding. Fortherapeutic or diagnostic purposes the main consideration is that theantibody must bind more strongly to one or more types of CCR4⁺ cellsthan to any CCR4⁻ with which the antibody may come into contact duringthe therapeutic or diagnostic application.

The antibody of the invention may be referred to as “CCR4-specific”. Theterm “CCR-specific” should be interpreted such that the binding of theantibody to CCR4 expressing cells is specific enough to allow the use ofsaid antibody for therapeutic or diagnostic purposes. The skilled personcan easily determine if any given antibody is CCR4-specific by comparingthe binding strength to the target CCR4⁺ cell with the binding strengthto one or more types of CCR4⁻ cells, e.g. wild-type (i.e. nottransformed with CCR4) HEK293T-cells or DT40-cells.

The skilled person will be aware that binding to CCR4⁺ cells compared toCCR4⁻ cells may be assessed, for example, using flow cytometry and asuitable example is described in Example 2.

Immunohistochemistry techniques, which are well known in the art, may beused to score the binding of antibodies to cells or samples. Such assaysmay be used to test the specificity of a particular antibody, or todetect CCR4 expression in tissue samples. Briefly, the antibody maytested for example on a high-density array of human tissues including apositive control (cells known to be CCR4-positive) and a negativecontrail (cells known to be CCR4-negative). The membranous stainingintensity may be estimated by visual inspection in a four step scale(0,1,2,3). Preferred antibodies show weak or strong, preferably strongimmunohistochemical scores for CCR4+ tissues.

Species cross-reactivity may be assayed using known methods and asuitable assay is described in Example 8.

The antibodies 17G, 9E, 1O and 11F have been shown to be capable ofinhibiting the binding of CCR4 to its ligands TARC and MDC (Example 3).Thus, preferably the antibodies of the invention are capable ofinhibiting the binding of CCR4 to one or more of its ligands.Preferably, the binding to at least MDC is inhibited. More preferably,the binding to MDC and TARC is inhibited. In some embodiments, thebinding of CCR4 to TARC is inhibited. In embodiments of any of theaspects disclosed herein, the antibodies of the invention are capable ofinhibiting the binding of MDC and/or TARC to CCR4. Thus, althoughreference is made throughout this text to the inhibition of the bindingof MDC to CCR4, an embodiment of any of the aspects and embodimentsdisclosed herein is the inhibition of binding to MDC and/or TARC.

By the “inhibition of binding” of a ligand to CCR4 is meant that bindingof the ligand to CCR4 is reduced by at least 20, 30, or 40%, morepreferably at least 45, 50, 55, 60, 65, 70 or 75%, even more preferablyat least 80% in the presence of the antibody compared to binding in theabsence of the antibody. Embodiments in which the binding of ligand toCCR4 is reduced by at least 85, 90 or 95% are also contemplated.Alternatively viewed, when the ligand is first contacted with CCR4 andthe antibody is subsequently added, the ligand can inhibit the bindingof the antibody to CCR4.

Assays for determining whether an antibody can inhibit the binding of aligand to CCR4 are well known and a suitable assay is descried inExample 3. Briefly, CCR4+ cells were incubated with MDC or without MDC,then antibody was added and the antibody was then detected with labeledanti-human antibody. Pre-incubation in the presence of MDC resulted in areduction in antibody binding to CCR4. Particularly, the binding ofantibody to DT40 cells (ATCC CRL-2111) transfected with CCR4 andpre-incubated with MDC or TARC is inhibited.

Alternative assays for determining whether an antibody can block thebinding of a ligand to CCR4 include the use of labeled ligand, e.g.radiolabelled ligand. A suitable assay is described in Example 9.

The antibodies 17G and 9E have been shown to be capable of inhibitingCCR4-mediated cellular responses to a CCR4 ligand, in particular byinhibiting the increase in intracellular calcium ion concentration inresponse to a CCR4 ligand (see Example 5). Dose-dependent inhibition ofTARC-induced signalling was demonstrated for antibodies 9E and 9E10J(see Example 5). Thus, the antibodies of the invention are preferablycapable of inhibiting CCR4-mediated cellular responses to a CCR4 ligand,in particular by inhibiting the increase in intracellular calcium ionconcentration in response to a CCR4 ligand. In particular, theantibodies are preferably capable of inhibiting MDC-induced calcium fluxand/or TARC-induced calcium flux in CCRF-CEM cells (ATCC CCL-119).Suitable assay methods are known and one assay is disclosed in Example5.

Other preferred properties include the absence of significant toxicityin vivo when the antibodies of the invention are administered and theabsence of significant other side effects in vivo.

In some embodiments, the antibodies may inhibit chemotaxis of CCR4+cells towards a ligand of CCR4 such as MDC or TARC. Antibodies 9E and9E10J were shown to be able to inhibit chemotaxis of CCR4+ cells towardsa ligand of CCR4 (Example 13). Thus, the antibodies of the invention arepreferably capable of inhibit chemotaxis of CCR4+ cells towards a ligandof CCR4, preferably MDC and/or TARC.

In some embodiments, the antibodies may induce complement-dependentcytotoxicity (CDC) of CCR4+ cells, but in other embodiments theantibodies are not capable of inducing CDC. In some embodiments, theantibodies may induce apoptosis of CCR4+ cells, but in other embodimentsthe antibodies are not capable of inducing apoptosis. Antibody 9E wasshown not to induce apoptosis of Ramos cells (see Example 12), so theantibodies of the invention are in some embodiments not capable ofinducing significant apoptosis of CCR4+ cells. In some embodiments, theantibodies may be internalised by CCR4+ cells upon binding to CCR4, butin other embodiments no significant internalisation takes place.

The induction of apoptosis may be assayed using well-known standardmethods, for example methods which assay Annexin V staining. Briefly,cells may be incubated with an antibody for a suitable period of time,e.g. 24 hours and the effect, after cell harvesting and Annexin Vstaining may be measured by FACS analysis (e.g. using EasyCyte).

The induction of CDC may be assayed using well-known standard methods,for example methods which measure the relative number of viable cellsbased on the uptake and metabolism of a redox dye such as Alamar blue. Asuitable assay is disclosed in H Gazzano-Santoro et al. J ImmunolMethods. 1997, 28; 202(2):163-71.

The skilled person will be aware of suitable ways to assayinternalisation, for example using temperature-differential fluorescencelabeling on flow cytometry or confocal microscopy. An example of asuitable assay involves a secondary antibody labelled with apH-sensitive dye (such as CypHer5E), which is minimally fluorescent at abasic pH (as found outside of cells) and maximally fluorescent at anacidic pH (as found inside of cells).

The inhibition of chemotaxis may be assayed using standard methods, forexample using a transwell assay. Briefly, cells capable of chemotaxisand which express CCR4 are contacted with an antibody in one chamber anda ligand of CCR4 such as MDC is placed in another chamber separated fromthe first chamber by a membrane of filter having a suitable pore size.The effect of the antibody on cell migration towards the ligand(chemotaxis) is determined by comparing chemotaxis in the presence ofthe antibody to chemotaxis in the absence of the antibody.

The term “ligand” of CCR4 includes the natural ligands of CCR4 such asMDC, TARC, RANTES, MCP-1 and/or MIP-1alpha, which may be naturallyproduced, recombinantly expressed or synthesised in the laboratory.

By “CCR4⁺ cells” is meant cells which express CCR4 on their surface,preferably at least substantially in its wild-type conformation. CCR4+cells may be naturally positive for CCR4, or they may be transformantswhich express recombinant CCR4.

In light of this invention, therefore, a range of anti-CCR4 antibodiescan be made and used in a variety of embodiments, including in thetreatment of any of the disorders discussed elsewhere herein,particularly cancer, immune disorders, inflammatory disorders andinfections.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore, an “antibody”, as used herein, means “at least afirst antibody”. The operable limits and parameters of combinations, aswith the amounts of any single agent, will be known to those of ordinaryskill in the art in light of the present disclosure.

Preferred embodiments of the invention are compositions comprising atleast one anti-CCR4 antibody of the invention, or antigen bindingfragment thereof.

Nucleic acid molecules comprising nucleotide sequences that encode theantibodies of the present invention as defined herein or parts orfragments thereof, or nucleic acid molecules substantially homologousthereto, form yet further aspects of the invention. Preferred nucleicacid molecules comprise sequences which encode the amino acid sequenceset out in SEQ ID NO: 35 (which is preferably encoded by SEQ ID NO: 34),SEQ ID NO: 46 (which is preferably encoded by SEQ ID NO: 45), SEQ ID NO:104 (which is preferably encoded by SEQ ID NO: 103), SEQ ID NO: 114(which is preferably encoded by SEQ ID NO: 113), SEQ ID NO: 57 (which ispreferably encoded by SEQ ID NO: 56) or SEQ ID NO: 68 (which ispreferably encoded by SEQ ID NO: 67). Other preferred nucleic acidmolecules comprise sequences which encode a heavy chain variable region(VH) that has the amino acid sequence of SEQ ID NO: 69, 71, 73, 75 or105 (which is preferably encoded by SEQ ID NO: 77, 79, 81, 83 or 106respectively) or 139, and/or comprise sequences which encode a lightchain variable region (VL) which has the amino acid sequence of SEQ IDNO: 70, 72, 74, 76 or 115 (which is preferably encoded by SEQ ID NO: 78,80, 82, 84 or 116 respectively) or 140. More preferred are nucleic acidswhich encode the following combinations: SEQ ID NOs: 139 and 140; or SEQID NOs: 69 and 70; or SEQ ID NOs: 71 and 72; or SEQ ID NOs 105 and 72;or SEQ ID NOs 71 and 115; or SEQ ID NOs 73 and 74; or SEQ ID NOs 75 and76. Also preferred are nucleic acid molecules which comprise thefollowing combinations: SEQ ID NOs: 77 and 78; or SEQ ID NOs: 79 and 80;or SEQ ID NOs: 106 and 80; or SEQ ID NOs: 79 and 116; or SEQ ID NOs: 81and 82; or SEQ ID NOs: 83 and 84.

Other preferred nucleic acid molecules comprise sequences that encodeIgG forms of the antibodies of the invention, for example those asdescribed in Example 1, or murine chimeric forms.

As indicated above, other nucleic acid molecules encompassed by thepresent invention are those encoding parts or fragments of the humanantibodies of the present invention, e.g., those encoding a heavy chainvariable region (VH) of an antibody (e.g., those encoding SEQ ID NO: 69,71, 73, 75 or 105, such as SEQ ID NOs: 77, 79, 81, 83 or 106respectively) or those encoding a light chain variable region (VL) of anantibody (e.g., those encoding SEQ ID NO: 70, 72, 74, 76 or 115, such asSEQ ID NO: 78, 80, 82, 84 or 116 respectively). Other preferred nucleicacid molecules are those encoding a heavy chain of an antibody of thepresent invention (e.g., those encoding SEQ ID NO:87, 91, 95, 99, 119 or123, such as SEQ ID NOs: 85, 89, 93, 97, 117 or 121 respectively) orthose encoding a light chain of an antibody (e.g., those encoding SEQ IDNO: 88, 92, 96, 100, 120 or 124 such as SEQ ID NOs: 86, 90, 94, 98, 118or 122 respectively).

Thus, fragments of the antibodies of the invention as defined herein, orsequences substantially homologous thereto, or nucleic acid moleculescomprising sequences encoding such fragments form a yet further aspectof the invention.

Advantageously, the antibodies of the present invention, when in IgGformat, have a high binding affinity for CCR4, i.e., have a Kd in therange of 1×10⁻⁸ M or 1×10⁻⁹ M or less. Importantly, antibodies with suchan affinity are in the established range that has been shown to beuseful for therapy. Preferably, the antibodies of the invention, when inIgG format, have a binding affinity for CCR4 that corresponds to a Kd ofless than 30 nM, 20 nM, 15 nM or 10 nM, more preferably of less than 10,9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1nM, most preferably less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or0.1 nM.

Any appropriate method of determining Kd may be used. However,preferably the Kd is determined by testing various concentrations of thetest antibody against various concentrations of antigen (CCR4) in vitroto establish a saturation curve, for example using the Lineweaver-Burkmethod, or by using commercially available binding model software, suchas the 1:1 binding model in the BIAcore 1000 Evaluation software. Asuitable assay in which Kd values were calculated from IgG titrations onCCR+ cells using “one site-specific binding” model f the software Prism(GraphPad, San Diego, Calif.) is used in Example 2.

With regard to determinations of Kd values, the skilled person willappreciate that apparent Kd values derived from binding experimentsusing cells expressing a target (e.g. CCR4) cannot be considered to bean absolute indication of affinity, because the experimental conditionswill affect the apparent binding affinity. For example, the levels ofexpression of CCR4 may vary depending on the conditions under which thecells are cultured, as well as differing between different cell types.It is consequently best to compare apparent Kd values obtained withinone set of experiments and it may not always be appropriate to compareKd values obtained in one set of experiments with Kd values obtained ina different set of experiments, particularly if the experimentalconditions varied significantly.

Alternatively, the off-rate and the antibody half-life on the surface ofthe CCR4-positive cell can be determined by performing the cell surfaceretention assay Adams et al., 1998, Prolonged in vivo tumour retentionof a human diabody targeting the extracellular domain of human HER2/neu.Br J Cancer 77: 1405-12; Le Gall et al., 1999, Di-, tri- and tetramericsingle chain Fv antibody fragments against human CD19: effect of valencyon cell binding. FEBS Lett 453: 164-8. The latter method allows moreappropriate mimicking the real situation in human patient under thetreatment conditions.

In some embodiments, antibodies of the invention may bind to both humanCCR4 and monkey CCR4. Such cross-reactivity between species and inparticular between humans and species commonly used as pre-clinicalanimal models may be an advantage as it allows a more effectivetranslation from pre-clinical studies to clinical use. For example,having an antibody which cross reacts with the native CCR4 present inthe particular animal model used means that the results in this modelare more likely to reflect the situation in a human patient, therebyallowing a more accurate assessment of for example dosing to be made andan increased likelihood of identifying any potentially relevant orproblematic side effects. For example, the ability of an antibody of theinvention to bind to both human CCR4 and monkey CCR4 means that suchantibodies can be tested in preclinical toxicity studies to assessadverse side effects of the treatment and to find appropriate tolerateddosages.

In addition, the ability to bind both human CCR4 and mouse CCR4 meansthat the results shown by such antibodies of the invention in mousemodels, e.g. mouse syngeneic models using immunocompetent mice, are morelikely to be representative of the activity of the antibodies in humansubjects. The reason for this is that antibodies which can bind to humanCCR4 but not mouse CCR4 will bind to CCR4 expressed by the human tumorcells in the mouse model but will not be able to bind to endogenousmurine CCR4. This is of course unlike the situation in a human patient,in which CCR4 expressed by the tumor and endogenous CCR4 would bepresent.

This is especially the case if the antibody has similar affinity to bothmurine and human CCR4.

The potential disadvantage with such a situation is that an antibodywhich binds to human CCR4 but not, or with significantly lower affinity,to mouse CCR4 might perform well in a human tumor xenograft model inimmunocompromized mice (e.g. nude or SCID mice) but this might not bereflected by a similar performance in a human system where much moreCCR4 was present. In other words, the anti-tumor effect seen in a mousexenograft system with an antibody which can bind to human CCR4 but notmouse CCR4 might look better than the clinical reality. In contrast,when working with an antibody that can bind to both human and mouse CCR4then this will bind to all forms of CCR4 present in the mouse modelsystem and is likely to be more representative of the situation when theantibody is put into humans. This is especially the case if the antibodyhas similar affinity to both murine and human CCR4.

In preferred embodiments, antibodies of the invention bind to human andmonkey CCR4 or to human and mouse CCR4 with similar affinities, e.g.with a Kd of 10 nM or less or 5 nM or less, more preferably 3 nM or lessor 2 nM or less, most preferably 1 nM or less.

By “similar affinity” is also meant that the binding affinity of theantibody for human CCR4 and for one or more of the other species ofinterest (e.g. monkey or mouse) is comparable, e.g. is not more than afactor of 20 different. More preferably the difference between thebinding affinities is less than a factor of 15, more preferably lessthan a factor of 10, most preferably less than a factor of 5, 4, 3 or 2.

However, in other embodiments the antibodies of the present inventionmay not bind to monkey CCR4 and/or they may not bind to mouse CCR4.

The antibodies of the invention bind to CCR4. Thus the antibodies orbinding proteins of the invention can be used to detect CCR4 in vivo orin vitro, in particular to detect CCR4+ cells. For example, as CCR4 isexpressed on certain tumour cells, the antibodies or binding proteins ofthe invention can be used to detect tumour cells in vivo or in vitro. Inaddition, the ability of the antibodies to localize to CCR4+ cells meansthat the antibodies of the invention can target body sites at whichCCR4+ cells are present, whereupon the antibody can act at the targetsite. In particular, the ability of the antibodies to localize to CCR4+tumour cells means that the antibodies of the invention can target bodysites at which CCR4+ tumour cells are present, whereupon the antibodycan act at the target site.

For example, the antibody may induce an anti-CCR4+ cell effect itselfi.e. as a naked antibody, e.g. by activating or inducing ADCC. Thisability to act as a naked Ab is advantageous. Alternatively, or inaddition, the antibody can induce an anti-CCR4+ cell effect by virtue ofbeing conjugated to an additional therapeutic molecule, e.g. a toxin orother anti-cancer molecule or an anti-inflammatory agent as describedherein.

The antibodies of the invention preferably have the ability to induceantibody dependent cellular cytotoxicity (ADCC) of CCR4+ cells. ADCC maybe assayed in vitro using methods well known in the art. A suitablemethod is described in Example 6. Alternatively, a Chromium-51 releaseassay may be used, for example. Thus, the antibodies of the inventionmay for example cause at least 10%, 15%, 20%, 22%, 25%, 30%, 40%, 50%,60%, 70%, 80% or 90% killing of CCR4+ cells in vitro e.g., in thepresence of human PBMCs. For example, the antibody 17G has been shown tocause at least 10%, 15%, 20% or 22% killing of the CCR4+ cell lineCCRF-CEM in the presence of human PBMCs and the antibody 9E has beenshown to cause at least 10%, 15%, 20%, 22%, 30%, 40%, 45%, 50%, 55%killing of the CCR4+ cell line CCRF-CEM in the presence of human PBMCs(see Example 6). Antibody 9E10J has been shown to cause at least 10%,15%, 20%, 22%, 30%, 35%, 40%, 42% killing of the CCR4+ cell lineCCRF-CEM in the presence of human PBMCs (see Example 6). ADCC isadvantageous for some applications, particularly some therapeuticapplications. Thus, in preferred embodiments the antibody can induceADCC of CCR4+ cells, preferably of CCR4+ tumour cells and/or CCR4+ Th2cells. In some embodiments the antibody-mediated ADCC is in the presenceof PBMCs, but embodiments in which antibody-mediated ADCC is in theabsence of PBMCs are also contemplated. In other embodiments, theantibodies induce little or no significant ADCC.

The antibodies of the invention are preferably also shown to be suitablypotent in terms of the concentration of antibody required to achievesuch ADCC levels. Again, a suitable in vitro test is described inExample 6. Thus, the antibody concentration required for half maximalcell lysis (EC₅₀) of CCR4+ cells, e.g. CCRF-CEM cells, in vitro ispreferably less than 700 ng/ml, 650 ng/ml, 620 ng/ml, 600 ng/ml, 550ng/ml, 500 ng/ml, 450 ng/ml, 400 ng/ml, 350 ng/ml, 300 ng/ml, 250 ng/ml,200 ng/ml, 150 ng/ml, 100 ng/ml, 90 ng/ml, 80 ng/ml, 70 ng/ml, 60 ng/ml,50 ng/ml, 46 ng/ml, 40 ng/ml, 35 ng/ml, 30 ng/ml, 25 ng/ml, 20 ng/ml, 15ng/ml, 10 ng/ml, 9 ng/ml, 7 ng/ml, 5 ng/ml, 2 ng/ml, 1 ng/ml, 0.5 ng/mlor 0.25 ng/ml. For example, the 17G antibody of the invention has beenshown to have an EC₅₀ of 619 ng/ml for CCRF-CEM cells, and antibody 9Eof the invention has been shown to have an EC₅₀ of 46 ng/ml for CCRF-CEMcells and in a separate experiment antibody 9E10J of the invention hasbeen shown to have an EC₅₀ of 25 ng/ml for CCRF-CEM cells (see Example6).

Preferably, the above described abilities are observed at a measurableor significant level and more preferably at a statistically significantlevel, when compared to appropriate control levels.

It should be noted that PBMC (effector cells) prepared from differentdonors may exhibit a significant variation of ADCC with respect to theextent of non-specific and specific tumour cell lysis, as well as EC50values. This phenomenon has been described by Naundorf et al, 2002 andit was also observed when assaying ADCC by the antibodies of the presentinvention (see Examples 6 and 11).

Human IgG1 is a glycoprotein bearing two N-linked oligosaccharide chainsbound to the Fc region. The oligosaccharides are of the complexbiantennary type, composed of a trimannosyl core structure with thepresence or absence of core fucose, bisecting N-acetylglucosamine(GlcNAc), galactose, and terminal sialic acid, which gives rise tostructural heterogeneity. Both human serum IgG and therapeuticantibodies are well known typically to be heavily fucosylated.

It has been reported that ADCC enhancement may in some instances beachieved by manipulating the state of oligosaccharides on human IgG1subclass. In particular, defucosylation has been shown to cause anincrease in ADCC activity of some antibodies (Niwa R et al, 2004). Thus,in preferred embodiments, antibodies according of the invention aremodified during production/expression of the protein, and/or in vitroafter production/expression, to generate a specific glycosylationpattern, particularly a glycosylation pattern which is beneficial fortherapeutic application of the antibodies. Preferably, said specificglycosylation pattern is the reduction or absence of fucose-basedglycosylation, which preferably increases the antibody's ability toinduce ADCC. Thus, in preferred embodiments, the antibodies of theinvention have a specific glycosylation pattern, preferably a specificglycosylation pattern which increases the ability of said antibody toinduce ADCC. Preferable, the antibodies of the invention aredefucosylated or non-fucosylated.

The skilled person is aware of suitable ways of preparing defucosylatedor non-fucosylated antibodies. As described in Example 10, this can beachieved by producing the antibody in presence of Kifunensine (forexample 100 ng/ml), a selective inhibitor of class I α-mannosidases,leading to a decrease in fucosylation of the molecule during production.Suitable host cells which lack one or more proteins required forfucosylation of oligosaccharide moieties can be used to producedefucosulated antibodies, e.g. fucosyltransferase-deficient host cells.Examples of suitable host cells are cells wherein the activity of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose and/or the activity of an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through an α-bond in the complexN-glycoside-linked sugar chain is decreased or deleted. Examples of suchenzymes include enzymes relating to the synthesis of GDP-fucose includeGMD (GDP-mannose 4,6-dehydratase), Fx (GDP-keto-6-deoxymannose3,5-epimerase, 4-reductase), GFPP (GDP-beta-L-fucose pyrophosphorylase).

By “defucosulated” is meant that at least 10%, preferably at least 20,30, 40 50, 60, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or atleast 99% of the total complex N-glycoside-linked sugar chains bound tothe Fc region are sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain.

By “non-fucosylated” is meant that no significant levels of fucose arepresent in the antibody.

Some antibodies are capable of being internalized into the cells towhich they become bound. Thus, in some embodiments of the invention theantibodies are capable of being internalized. This property isparticularly advantageous for use in immunoconjugates as any other agentattached to the antibody molecule should be internalized with theantibody molecule. In other embodiments no significant internalizationis seen.

Particularly for medical applications, it is desirable that the andibodydoes not induce any significant platelet aggregation. Plateletaggregation may be assayed as described in Example 15. This Exampleshows that antibody 9E10J does not have any significant effect onplatelet aggregation. Thus, in some embodiments of the invention theantibodies do not have any significant effect on platelet aggregation.

As discussed above, certain PBLs, including Tregs, express CCR4, so theability of antibody 9E10J to PBLs was assayed in Example 16 and thisExample shows that 9E10J can bind PBLs. Thus, in some embodiments of theinvention the antibodies can bind to PBLs, preferably to Tregs and/orTh2 cells. This feature is advantageous, particularly in immunotherapy,as it may allow the depletion of Treg cells.

In the following descriptions of the compositions, immunoconjugates,pharmaceuticals, combinations, cocktails, kits, first and second medicaluses and all methods in accordance with this invention, the terms“antibody” and “immunoconjugate”, or an antigen-binding region orfragment thereof, unless otherwise specifically stated or made clearfrom the scientific terminology, refer to a range of anti-CCR4antibodies as well as to the specific 17G, 9E, 1O, 11F, 9E10J and 9E1 Dantibodies.

The terms “antibody” and “immunoglobulin”, as used herein, refer broadlyto any immunological binding agent or molecule that comprises a humanantigen binding domain, including polyclonal and monoclonal antibodies.Depending on the type of constant domain in the heavy chains, wholeantibodies are assigned to one of five major classes: IgA, IgD, IgE,IgG, and IgM and the antibodies of the invention may be in any one ofthese classes. Several of these are further divided into subclasses orisotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chainconstant domains that correspond to the difference classes ofimmunoglobulins are termed α, δ, ε, γ and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regionsare used in the invention, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting. IgG1 antibodies areparticularly preferred.

The “light chains” of mammalian antibodies are assigned to one of twoclearly distinct types: kappa (κ) and lambda (λ), based on the aminoacid sequences of their constant domains and some amino acids in theframework regions of their variable domains. There is essentially nopreference to the use of κ or λ light chain constant regions in theantibodies of the present invention.

As will be understood by those in the art, the immunological bindingreagents encompassed by the term “antibody” extend to all antibodies andantigen binding fragments thereof, including whole antibodies, dimeric,trimeric and multimeric antibodies; bispecific antibodies; chimericantibodies; recombinant and engineered antibodies, and fragmentsthereof.

The term “antibody” is thus used to refer to any antibody-like moleculethat has an antigen binding region, and this term includes antibodyfragments that comprise an antigen binding domain such as Fab′, Fab,F(ab′)₂, single domain antibodies (DABs), T and Abs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fabfusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); Bispecific T-cell Engager (BiTE)(scFv-scFv tandems to attract T cells); dual variable domain (DVD)-Ig(bispecific format); small immunoprotein (SIP) (kind of minibody); SMIP(“small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilizeddiabody “Dual Affinity ReTargeting”); small antibody mimetics comprisingone or more CDRs and the like.

The techniques for preparing and using various antibody-based constructsand fragments are well known in the art (see Kabat et al., 1991,specifically incorporated herein by reference). Diabodies, inparticular, are further described in EP 404, 097 and WO 93/11161;whereas linear antibodies are further described in Zapata et al. (1995).

Antibodies can be fragmented using conventional techniques. For example,F(ab′)₂ fragments can be generated by treating the antibody with pepsin.The resulting F(ab′)₂ fragment can be treated to reduce disulfidebridges to produce Fab′ fragments. Papain digestion can lead to theformation of Fab fragments. Fab, Fab′ and F(ab′)₂, scFv, Fv, dsFv, Fd,dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques or can be chemically synthesized. Techniques forproducing antibody fragments are well known and described in the art.For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; LeGall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young etal., 1995 further describe and enable the production of effectiveantibody fragments.

The antibodies or antibody fragments can be produced naturally or can bewholly or partially synthetically produced. Thus the antibody may befrom any appropriate source, for example recombinant sources and/orproduced in transgenic animals or transgenic plants, or in eggs usingthe IgY technology. Thus, the antibody molecules can be produced invitro or in vivo.

Preferably, the antibody or antibody fragment comprises an antibodylight chain variable region (V_(L)) that comprises three CDR domains andan antibody heavy chain variable region (V_(H)) that comprises three CDRdomains. Said VL and VH generally form the antigen binding site.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region has a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions (CDRs) conferantigen-binding specificity to the antibody.

However, it is well documented in the art that the presence of threeCDRs from the light chain variable domain and three CDRs from the heavychain variable domain of an antibody is not necessary for antigenbinding. Thus, constructs smaller than the above classical antibodyfragment are known to be effective.

For example, camelid antibodies (Hamers-Casterman et al., 1993; ArbabiGhahroudi et al., 1997) have an extensive antigen binding repertoire butare devoid of light chains. Also, results with single domain antibodiescomprising VH domains alone (Ward et al., 1989; Davies and Riechmann,1995) or VL domains alone (van den Beucken et al., 2001) show that thesedomains can bind to antigen with acceptably high affinities. Thus, threeCDRs can effectively bind antigen.

It is also known that a single CDR, or two CDRs, can effectively bindantigen. As a first example, a single CDR can be inserted into aheterologous protein and confer antigen binding ability on theheterologous protein, as exemplified by showing that a VH CDR3 regioninserted into a heterologous protein, such as GFP, confers antigenbinding ability on the heterologous protein (Kiss et al., 2006; Nicaiseet al., 2004).

It is further known that two CDRs can effectively bind antigen, and evenconfer superior properties than possessed by the parent antibody. Forexample, it has been shown (Qiu et al., 2007) that two CDRs from aparent antibody (a VH CDR1 and a VL CDR3 region) retain the antigenrecognition properties of the parent molecule but have a superiorcapacity to penetrate tumours. Joining these CDR domains with anappropriate linker sequence (e.g., from VH FR2) to orientate the CDRs ina manner resembling the native parent antibody produced even betterantigen recognition. Therefore, it is known in the art that it ispossible to construct antigen binding antibody mimetics comprising twoCDR domains (preferably one from a VH domain and one from a VL domain,more preferably, with one of the two CDR domains being a CDR3 domain)orientated by means of an appropriate framework region to maintain theconformation found in the parent antibody.

Thus, although preferred antibodies of the invention might comprise sixCDR regions (three from a light chain and three from a heavy chain),antibodies with fewer than six CDR regions and as few as one or two CDRregions are encompassed by the invention. In addition, antibodies withCDRs from only the heavy chain or light chain are also contemplated.

Preferred antibodies of the invention that bind to CCR4 comprise atleast one heavy chain variable region that comprises three CDRs and atleast one light chain variable region that comprises three CDRs, whereinsaid heavy chain variable region comprises:

-   -   (a) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO: 1 or a sequence substantially homologous thereto,    -   (b) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2        or a sequence substantially homologous thereto, and    -   (c) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3        or 15 or a sequence substantially homologous thereto; or    -   (d) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO: 7 or 101 or a sequence substantially homologous        thereto,    -   (e) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 8        or a sequence substantially homologous thereto, and    -   (f) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9        or a sequence substantially homologous thereto; or    -   (g) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO: 19 or a sequence substantially homologous thereto,    -   (h) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 20        or a sequence substantially homologous thereto, and    -   (i) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 21        or a sequence substantially homologous thereto; or    -   (j) a variable heavy (VH) CDR1 that has the amino acid sequence        of SEQ ID NO: 125 or 126 or a sequence substantially homologous        thereto,    -   (k) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 127        or 128 or a sequence substantially homologous thereto, and    -   (l) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 9        or a sequence substantially homologous thereto.

Preferred light chain CDR regions for use in conjunction with thespecified heavy chain CDR regions are described elsewhere herein.However, other light chain variable regions that comprise three CDRs foruse in conjunction with the heavy chain variable regions of theinvention are also contemplated. Appropriate light chain variableregions which can be used in combination with the heavy chain variableregions of the invention and which give rise to an antibody which bindsCCR4 can be readily identified by a person skilled in the art.

For example, a heavy chain variable region of the invention can becombined with a single light chain variable region or a repertoire oflight chain variable regions and the resulting antibodies tested forbinding to CCR4. It would be expected that a reasonable number of suchcombinations of heavy chain variable regions of the invention withdifferent light chain variable regions would retain the ability to bindCCR4.

Similar methods could be used to identify alternative heavy chainvariable regions for use in combination with preferred light chainvariable regions of the invention.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region, or aportion thereof. Furthermore, the antibody or antibody fragment cancomprise all or a portion of a kappa light chain constant region or alambda light chain constant region, or a portion thereof. All or part ofsuch constant regions may be produced naturally or may be wholly orpartially synthetic. Appropriate sequences for such constant regions arewell known and documented in the art. When a full complement of constantregions from the heavy and light chains are included in the antibodiesof the invention, such antibodies are typically referred to herein as“full length” antibodies or “whole” antibodies.

Antibodies containing an Fc region are preferred for certain uses,particularly therapeutic uses in vivo, where the Fc region mediateseffector functions such as ADCC.

The term “substantially homologous” as used herein in connection with anamino acid or nucleic acid sequence includes sequences having at least70% or 75%, preferably at least 80%, and even more preferably at least85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acidor nucleic acid sequence disclosed. Substantially homologous sequencesof the invention thus include single or multiple base or amino acidalterations (additions, substitutions, insertions or deletions) to thesequences of the invention. At the amino acid level preferredsubstantially homologous sequences contain only up to 1, 2, 3, 4 or 5,preferably up to 1, 2 or 3, more preferably up to 1 or 2, altered aminoacids, in one or more of the framework regions and/or one or more of theCDRs making up the sequences of the invention. Said alterations can bewith conservative or non-conservative amino acids. Preferably saidalterations are conservative amino acid substitutions.

The substantially homologous nucleic acid sequences also includenucleotide sequences that hybridize to the nucleic acid sequencesdisclosed (or their complementary sequences), e.g., hybridize tonucleotide sequences encoding one or more of the light chain or heavychain CDRs of the invention, the light or heavy chain variable regionsof the invention, or the antibodies of the invention (or hybridize totheir complementary sequences), under at least moderately stringenthybridization conditions.

The term “substantially homologous” also includes modifications orchemical equivalents of the amino acid and nucleotide sequences of thepresent invention that perform substantially the same function as theproteins or nucleic acid molecules of the invention in substantially thesame way. For example, any substantially homologous antibody (or thesubstantially homologous nucleic acid encoding it) should retain theability to bind to CCR4 as described above. Preferably, anysubstantially homologous antibody should retain the functionalcapabilities of the antibody, e.g. as defined elsewhere herein.Preferably, any substantially homologous antibody should retain theability to specifically bind to the same epitope of CCR4 as recognizedby the antibody in question, for example, the same epitope recognized bythe CDR domains of the invention or the VH and VL domains of theinvention as described herein. Binding to the same epitope/antigen canbe readily tested by methods well known and described in the art, e.g.,using binding assays, e.g., a competition assay. Retention of otherfunctional properties can also readily be tested by methods well knownand described in the art.

Thus, a person skilled in the art will appreciate that binding assayscan be used to test whether “substantially homologous” antibodies havethe same binding specificities as the antibodies and antibody fragmentsof the invention, for example, binding assays such as ELISA assays orBIAcore assays can readily be used to establish whether such“substantially homologous” antibodies can bind to CCR4. As outlinedabove, a competition binding assay can be used to test whether“substantially homologous” antibodies retain the ability to specificallybind to substantially the same epitope of CCR4 as recognized by theantibodies of the invention. The method described below is only oneexample of a suitable competition assay. The skilled person will beaware of other suitable methods and variations.

Substantially homologous sequences of proteins of the invention include,without limitation, conservative amino acid substitutions, or forexample alterations that do not affect the VH, VL or CDR domains of theantibodies, e.g., include scFv antibodies where a different linkersequence is used or antibodies where tag sequences or other componentsare added that do not contribute to the binding of antigen, oralterations to convert one type or format of antibody molecule orfragment to another type or format of antibody molecule or fragment(e.g., conversion from Fab to scFv or vice versa), or the conversion ofan antibody molecule to a particular class or subclass of antibodymolecule (e.g., the conversion of an antibody molecule to IgG or asubclass thereof, e.g., IgG1 or IgG3).

A “conservative amino acid substitution”, as used herein, is one inwhich the amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Homology may be assessed by any convenient method. However, fordetermining the degree of homology between sequences, computer programsthat make multiple alignments of sequences are useful, for instanceClustal W (Thompson et al., 1994). If desired, the Clustal W algorithmcan be used together with BLOSUM 62 scoring matrix (Henikoff andHenikoff, 1992) and a gap opening penalty of 10 and gap extensionpenalty of 0.1, so that the highest order match is obtained between twosequences wherein at least 50% of the total length of one of thesequences is involved in the alignment. Other methods that may be usedto align sequences are the alignment method of Needleman and Wunsch(1970), as revised by Smith and Waterman (1981) so that the highestorder match is obtained between the two sequences and the number ofidentical amino acids is determined between the two sequences. Othermethods to calculate the percentage identity between two amino acidsequences are generally art recognized and include, for example, thosedescribed by Carillo and Lipton (1988) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.Programs that compare and align pairs of sequences, like ALIGN (Myersand Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) andgapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereuxet al., 1984) are also useful for this purpose. Furthermore, the Daliserver at the European Bioinformatics institute offers structure-basedalignments of protein sequences (Holm, 1993; 1995; 1998).

By way of providing a reference point, sequences according to thepresent invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% homology, sequence identity etc. may be determined using the ALIGNprogram with default parameters (for instance available on Internet atthe GENESTREAM network server, IGH, Montpellier, France).

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected that promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g., 20,25, 30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule, a1% mismatch may be assumed to result in about a 1° C. decrease in Tm.For example, if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm—5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. By way of further example, sequences that “hybridize” are thosesequences binding (hybridizing) under non-stringent conditions (e.g.,6×SSC, 50% formamide at room temperature) and washed under conditions oflow stringency (e.g., 2×SSC, room temperature, more preferably 2×SSC,42° C.) or conditions of higher stringency (e.g., 2×SSC, 65° C.) (whereSSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).

It is understood, however, that equivalent stringencies may be achievedusing alternative buffers, salts and temperatures. Additional guidanceregarding hybridization conditions may be found in: Current Protocols inMolecular Biology, John Wiley & Sons, N. Y., 1989, 6.3.1-6.3.6 and in:Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989, Vol. 3.

Generally speaking, sequences that hybridize under conditions of highstringency are preferred, as are sequences which, but for the degeneracyof the code, would hybridize under high stringency conditions.

In other preferred embodiments, second generation antibodies areprovided that have enhanced or superior properties in comparison to anoriginal anti-CCR4 antibody, such as 17G, 9E, 1O, 11F, 9E10J or 9E1 D.For example, the second generation antibodies may have a strongerbinding affinity for CCR4, a superior cross reactivity profile, superiorability to target CCR4+ cells, particularly tumour cells, an improvedability to induce ADCC, an improved ability to induce CDC, an improvedtreatment of the disorders discussed elsewhere herein.

Comparisons to identify effective second generation antibodies arereadily conducted and quantified, e.g., using one or more of the variousassays described in detail herein or in the art. Second generationantibodies that have an enhanced biological property or activity of atleast about 2-fold, 5-fold, 10-fold, 20-fold, and preferably, at leastabout 50-fold, in comparison to the anti-CCR4 antibodies of the presentinvention, as exemplified by the 17G, 9E, 1O, 11F, 9E10J or 9E1 Dantibody, are encompassed by the present invention.

The antibody, binding protein and nucleic acid molecules of theinvention are generally “isolated” or “purified” molecules insofar asthey are distinguished from any such components that may be present insitu within a human or animal body or a tissue sample derived from ahuman or animal body. The sequences may, however, correspond to or besubstantially homologous to sequences as found in a human or animalbody. Thus, the term “isolated” or “purified” as used herein inreference to nucleic acid molecules or sequences and proteins orpolypeptides, e.g., antibodies, refers to such molecules when isolatedfrom, purified from, or substantially free of their natural environment,e.g., isolated from or purified from the human or animal body (if indeedthey occur naturally), or refers to such molecules when produced by atechnical process, i.e., includes recombinant and synthetically producedmolecules.

Thus, when used in connection with a nucleic acid molecule, such termsmay refer to a nucleic acid substantially free of material with which itis naturally associated such as other nucleic acids/genes orpolypeptides. These terms may also refer to a nucleic acid substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or substantially free of chemical precursors, or otherchemicals when chemically synthesized. An isolated or purified nucleicacid may also be substantially free of sequences that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) from which the nucleic acid is derived or sequences thathave been made to flank the nucleic acid (e.g., tag sequences or othersequence that have no therapeutic value) by, for example, geneticengineering.

Thus, when used in connection with a protein or polypeptide moleculesuch as light chain CDRs 1, 2 and 3, heavy chain CDRs 1, 2 and 3, lightchain variable regions, heavy chain variable regions, and bindingproteins or antibodies of the invention, including full lengthantibodies, the term “isolated” or “purified” typically refers to aprotein substantially free of cellular material or other proteins fromthe source from which it is derived. In some embodiments, particularlywhere the protein is to be administered to humans or animals, suchisolated or purified proteins are substantially free of culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Such isolated or purifiedproteins may also be free of flanking sequences such as those describedabove for the isolated nucleic acid molecules.

The term “nucleic acid sequence” or “nucleic acid molecule” as usedherein refers to a sequence of nucleoside or nucleotide monomerscomposed of naturally occurring bases, sugars and intersugar (backbone)linkages. The term also includes modified or substituted sequencescomprising non-naturally occurring monomers or portions thereof. Thenucleic acid sequences of the present invention may be deoxyribonucleicacid sequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine. Thenucleic acid molecules may be double stranded or single stranded. Thenucleic acid molecules may be wholly or partially synthetic orrecombinant.

In preferred embodiments the antibodies of the invention are humanantibodies, more preferably fully human antibodies. In this regard,human antibodies generally have at least three potential advantages foruse in human therapy. First, the human immune system should notrecognize the antibody as foreign. Second, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given. Third, because theeffector portion is human, it will interact better with the other partsof the human immune system, e.g., to destroy target cells moreefficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC).

However, although human antibodies are generally recognized to displaythese advantages, it is known that the development of human antibodiesthat have high enough affinities and appropriate functional propertiesto make them candidates for successful human therapy is by no meansstraightforward. The art therefore still lacks anti-CCR4 for the safeand effective treatment of humans, and poses challenges to thedevelopment of such agents.

The term “human” as used herein in connection with antibody moleculesand binding proteins first refers to antibodies and binding proteinshaving variable regions (e.g., V_(H), V_(L), CDR or FR regions) and,optionally, constant antibody regions, isolated or derived from a humanrepertoire or derived from or corresponding to sequences found inhumans, e.g., in the human germline or somatic cells. The 17G, 9E, 1O,11F, 9E10J and 9E1 D antibodies are examples of such a human antibodymolecules wherein the variable regions have been isolated from a humanrepertoire.

The “human” antibodies and binding proteins of the invention furtherinclude amino acid residues not encoded by human sequences, e.g.,mutations introduced by random or site directed mutations in vitro, forexample mutations introduced by in vitro cloning or PCR. Particularexamples of such mutations are mutations that involve conservativesubstitutions or other mutations in a small number of residues of theantibody or binding protein, e.g., in up to 5, 4, 3, 2 or 1 of theresidues of the antibody or binding protein, preferably e.g., in up to5, 4, 3, 2 or 1 of the residues making up one or more of the CDRs of theantibody or binding protein. Certain examples of such “human” antibodiesinclude antibodies and variable regions that have been subjected tostandard modification techniques to reduce the amount of potentiallyimmunogenic sites.

Thus, the “human” antibodies of the invention include sequences derivedfrom and related to sequences found in humans, but which may notnaturally exist within the human antibody germline repertoire in vivo.In addition, the human antibodies and binding proteins of the presentinvention include proteins comprising human consensus sequencesidentified from human sequences, or sequences substantially homologousto human sequences.

In addition, the human antibodies and binding proteins of the presentinvention are not limited to combinations of V_(H), V_(L), CDR or FRregions that are themselves found in combination in human antibodymolecules. Thus, the human antibodies and binding proteins of theinvention can include or correspond to combinations of such regions thatdo not necessarily exist naturally in humans.

In preferred embodiments, the human antibodies will be fully humanantibodies. “Fully human” antibodies, as used herein, are antibodiescomprising “human” variable region domains and/or CDRs, as definedabove, without substantial non-human antibody sequences or without anynon-human antibody sequences. For example, antibodies comprising humanvariable region domains and/or CDRs “without substantial non-humanantibody sequences” are antibodies, domains and/or CDRs in which only upto 5, 4, 3, 2 or 1 amino acids are amino acids that are not encoded byhuman antibody sequences. Thus, “fully human” antibodies aredistinguished from “humanized” antibodies, which are based onsubstantially non-human variable region domains, e.g., mouse variableregion domains, in which certain amino acids have been changed to bettercorrespond with the amino acids typically present in human antibodies.

The “fully human” antibodies of the invention may be human variableregion domains and/or CDRs without any other substantial antibodysequences, such as being single chain antibodies. Alternatively, the“fully human” antibodies of the invention may be human variable regiondomains and/or CDRs integral with or operatively attached to one or morehuman antibody constant regions. Certain preferred fully humanantibodies are IgG antibodies with the full complement of IgG constantregions.

In other embodiments, “human” antibodies of the invention will bepart-human chimeric antibodies. “Part-human chimeric” antibodies, asused herein, are antibodies comprising “human” variable region domainsand/or CDRs operatively attached to, or grafted onto, a constant regionof a non-human species, such as rat or mouse. Such part-human chimericantibodies may be used, for example, in pre-clinical studies, whereinthe constant region will preferably be of the same species of animalused in the pre-clinical testing. These part-human chimeric antibodiesmay also be used, for example, in ex vivo diagnostics, wherein theconstant region of the non-human species may provide additional optionsfor antibody detection.

The term “fragment” as used herein refers to fragments of biologicalrelevance, e.g., fragments that contribute to antigen binding, e.g.,form part of the antigen binding site, and/or contribute to theinhibition or reduction in function of the CCR4 antigen. Certainpreferred fragments comprise a heavy chain variable region (V_(H)domain) and/or a light chain variable region (V_(L) domain) of theantibodies of the invention. Other preferred fragments comprise one ormore of the heavy chain CDRs of the antibodies of the invention (or ofthe V_(H) domains of the invention), or one or more of the light chainCDRs of the antibodies of the invention (or of the V_(L) domains of theinvention). Certain preferred fragments are at least 5 amino acids inlength and comprise at least one CDR region, preferably a CDR3 region,more preferably a heavy chain CDR3 region.

In embodiments where the antibodies of the invention comprise a fragmentof any of the defined sequences (for example comprise a fragment of SEQID NO:35, 46, 57, 68, 104 or 114), e.g., are antibodies comprising V_(H)and/or V_(L) domains of the invention, or are antibodies or bindingproteins comprising one or more CDRs of the invention, then theseregions/domains are generally separated within the antibody or bindingprotein so that each region/domain can perform its biological functionand so that the contribution to antigen binding is retained. Thus, theV_(H) and V_(L) domains are preferably separated by appropriate scaffoldsequences/linker sequences and the CDRs are preferably separated byappropriate framework regions such as those found in naturally occurringantibodies and/or effective engineered antibodies. Thus, the V_(H),V_(L) and individual CDR sequences of the invention are preferablyprovided within or incorporated into an appropriate framework orscaffold to enable antigen binding. Such framework sequences or regionsmay correspond to naturally occurring framework regions, FR1, FR2, FR3and/or FR4, as appropriate to form an appropriate scaffold, or maycorrespond to consensus framework regions, for example identified bycomparing various naturally occurring framework regions. Alternatively,non-antibody scaffolds or frameworks, e.g., T cell receptor frameworkscan be used.

Appropriate sequences that can be used for framework regions are wellknown and documented in the art and any of these may be used. Preferredsequences for framework regions are one or more (i.e. one, two, three orfour) of the framework regions making up the V_(H) and/or V_(L) domainsof the invention, i.e., one or more of the framework regions disclosedin Tables 1, 2, 3 or 4, or framework regions substantially homologousthereto, and in particular framework regions that allow the maintenanceof antigen specificity, for example framework regions that result insubstantially the same or the same 3D structure of the antibody.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs:30, 31, 32 and 33) and/or variable heavy chain (SEQ ID NOs:25, 26, 27 and 28), as appropriate, FR regions of SEQ ID NO: 35 (alsoshown in Table 1), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs:41, 42, 43 and 44) and/or variable heavy chain (SEQ ID NOs:36, 37, 38 and 39), as appropriate, FR regions of SEQ ID NO: 46 (alsoshown in Table 2), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs:41, 42, 43 and 44) and/or variable heavy chain (SEQ ID NOs:36, 37, 102 and 39), as appropriate, FR regions of SEQ ID NO: 103, or FRregions substantially homologous thereto, are found in the antibodies ofthe invention. Embodiments in which variable heavy chain FR region ofSEQ ID NO: 102 or a sequence substantially homologous thereto is presentare especially preferred.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 107, 109, 111 and 44) and/or variable heavy chain (SEQ IDNOs: 36, 37, 38 and 39), as appropriate, FR regions of SEQ ID NO: 114,or FR regions substantially homologous thereto, are found in theantibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs:52, 53, 54 and 55) and/or variable heavy chain (SEQ ID NOs:47, 48, 49 and 50), as appropriate, FR regions of SEQ ID NO: 57 (alsoshown in Table 3), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In certain preferred embodiments, all four of the variable light chain(SEQ ID NOs: 63, 64, 65 and 66) and/or variable heavy chain (SEQ ID NOs:58, 59, 60 and 61), as appropriate, FR regions of SEQ ID NO: 68 (alsoshown in Table 4), or FR regions substantially homologous thereto, arefound in the antibodies of the invention.

In addition, although preferred antibodies of the invention are made upof V_(H), V_(L) or CDRs of the invention, it should be noted that theantibodies of the invention also encompass one or more V_(H), V_(L) orCDRs of the invention in combination with other V_(H), V_(L) or CDRs notof the invention, provided that the CCR4 binding properties or anti-CCR4properties of the antibodies of the invention as outlined herein arestill present.

The term “heavy chain complementarity determining region” (“heavy chainCDR”) as used herein refers to regions of hypervariability within theheavy chain variable region (V_(H) domain) of an antibody molecule. Theheavy chain variable region has three CDRs termed heavy chain CDR1,heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxyterminus. The heavy chain variable region also has four frameworkregions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxyterminus). These framework regions separate the CDRs.

The term “heavy chain variable region” (V_(H) domain) as used hereinrefers to the variable region of a heavy chain of an antibody molecule.

The term “light chain complementarity determining region” (“light chainCDR”) as used herein refers to regions of hypervariability within thelight chain variable region (V_(L) domain) of an antibody molecule.Light chain variable regions have three CDRs termed light chain CDR1,light chain CDR2 and light chain CDR3 from the amino terminus to thecarboxy terminus. The light chain variable region also has fourframework regions (FR1, FR2, FR3 and FR4 from the amino terminus tocarboxy terminus). These framework regions separate the CDRs.

The term “light chain variable region” (V_(L) domain) as used hereinrefers to the variable region of a light chain of an antibody molecule.

It should be noted that the Kabat nomenclature is followed herein, wherenecessary, in order to define the positioning of the CDRs (Kabat et al.,1991, specifically incorporated herein by reference).

A person skilled in the art will appreciate that the proteins andpolypeptides of the invention, such as the light and heavy CDRs, thelight and heavy chain variable regions, antibodies, antibody fragments,and immunoconjugates, may be prepared in any of several ways well knownand described in the art, but are most preferably prepared usingrecombinant methods.

Nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention can be derived or produced byany appropriate method, e.g., by cloning or synthesis. Such sequencescould, for example, be prepared by cloning appropriate sequences frome.g., human germ line genes and then making any necessary modificationsto the germ line sequences to obtain the sequences of the inventionusing methods well known and described in the art. An alternative andmore efficient method would be to synthesize the appropriate light orheavy chain variable region sequence as overlapping primers, and useprimer extension to obtain the full sequence. This full sequence couldthen be amplified via PCR with primers containing appropriaterestriction sites for further cloning and manipulation, e.g., forcloning into an appropriate expression vector. Five to seven overlappingprimers per variable region are normally be sufficient, thereby makingthis technique very efficient and precise.

Once nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention have been obtained, thesefragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region fragments intofull length antibody molecules with appropriate constant region domains,or into particular formats of antibody fragment discussed elsewhereherein, e.g., Fab fragments, scFv fragments, etc. Typically, or as partof this further manipulation procedure, the nucleic acid fragmentsencoding the antibody molecules of the invention are generallyincorporated into an appropriate expression vector in order tofacilitate production of the antibodies of the invention.

Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which are operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner that allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vectorcontaining a nucleic acid molecule of the invention, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the protein sequence encoded by the nucleic acidmolecule of the invention.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (forexample, see the regulatory sequences described in Goeddel, 1990).Selection of appropriate regulatory sequences is dependent on the hostcell chosen as discussed below, and may be readily accomplished by oneof ordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asneomycin and hygromycin that confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes that encode afusion moiety that provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification (for example appropriate “tags” to enablepurification and/or identification may be present, e.g., His tags or myctags). For example, a proteolytic cleavage site may be added to thetarget recombinant protein to allow separation of the recombinantprotein from the fusion moiety subsequent to purification of the fusionprotein. Typical fusion expression vectors include pGEX (Amrad Corp.,Melbourne, Australia), pMal (New England Biolabs, Beverly, Mass.) andpRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g., a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofection, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al., 1989, and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, 1990. In addition, the proteins of theinvention may be expressed in prokaryotic cells, such as Escherichiacoli (Zhang et al., 2004).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari. et al., 1987), pMFa (Kurjan and Herskowitz,1982), pJRY88 (Schultz et al., 1987), and pYES2 (Invitrogen Corporation,San Diego, Calif.). Protocols for the transformation of yeast and fungiare well known to those of ordinary skill in the art (see Hinnen et al.,1978; Ito et al., 1983, and Cullen et al. 1987).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573), NS-1 cells, NS0 (ATCC CRL-11177), and Per.C6® (Crucell,Leiden, Netherlands). Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., 1987) and pMT2PC (Kaufman et al.,1987).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., 1987, which reviews the use ofAgrobacterium rhizogenes vectors; see also Zambryski et al., 1984, whichdescribes the use of expression vectors for plant cells, including,among others, PAPS2022, PAPS2023, and PAPS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., 1983)and the pVL series (Luckow and Summers 1989). Some baculovirus-insectcell expression systems suitable for expression of the recombinantproteins of the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. 1985; Palmiter et al. 1983; Brinster et al. 1985;Palmiter and Brinster 1985, and U.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield (1964); Frische et al., 1996) or synthesisin homogenous solution.

N-terminal or C-terminal fusion proteins comprising the antibodies andproteins of the invention conjugated to other molecules, such asproteins, may be prepared by fusing through recombinant techniques. Theresultant fusion proteins contain an antibody or protein of theinvention fused to the selected protein or marker protein, or tagprotein as described herein. The antibodies and proteins of theinvention may also be conjugated to other proteins by known techniques.For example, the proteins may be coupled using heterobifunctionalthiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins that may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Irrespective of the manner of preparation of a first anti-CCR4 antibodynucleic acid segment, further suitable antibody nucleic acid segmentsmay be readily prepared by standard molecular biological techniques. Inorder to confirm that any variant, mutant or second generation anti-CCR4antibody nucleic acid segment is suitable for use in the presentinvention, the nucleic acid segment will be tested to confirm expressionof an anti-CCR4 antibody in accordance with the present invention.Preferably, the variant, mutant or second generation nucleic acidsegment will also be tested to confirm hybridization under standard,more preferably, standard stringent hybridization conditions. Exemplarysuitable hybridization conditions include hybridization in about 7%sodium dodecyl sulfate (SDS), about 0.5 M NaPO₄, about 1 mM EDTA atabout 50° C.; and washing with about 1% SDS at about 42° C.

As a variety of antibodies may be readily prepared, the treatmentmethods of the invention may be executed by providing to the animal orpatient at least a first nucleic acid segment or molecule that expressesa biologically effective amount of at least a first anti-CCR4 antibodyof the invention in the patient. The “nucleic acid segment or moleculethat expresses an anti-CCR4 antibody” will generally be in the form ofat least an expression construct or vector, and may be in the form of anexpression construct or vector comprised within a virus or within arecombinant host cell. Preferred gene therapy vectors of the presentinvention will generally be viral vectors, such as comprised within arecombinant retrovirus, herpes simplex virus (HSV), adenovirus,adeno-associated virus (AAV), cytomegalovirus (CMV), and the like.

A yet further aspect provides an expression construct or expressionvector comprising one or more of the nucleic acid segments or moleculesof the invention. Preferably the expression constructs or vectors arerecombinant. Preferably said constructs or vectors further comprise thenecessary regulatory sequences for the transcription and translation ofthe protein sequence encoded by the nucleic acid molecule of theinvention.

A yet further aspect provides a host cell or virus comprising one ormore expression constructs or expression vectors of the invention. Alsoprovided are host cells or viruses comprising one or more of the nucleicacid molecules of the invention. A host cell or virus expressing anantibody of the invention forms a yet further aspect.

A yet further aspect of the invention provides a method of producing anantibody of the present invention comprising a step of culturing thehost cells of the invention. Preferred methods comprise the steps of (i)culturing a host cell comprising one or more of the recombinantexpression vectors or one or more of the nucleic acid sequences of theinvention under conditions suitable for the expression of the encodedantibody or protein; and optionally (ii) isolating or obtaining theantibody or protein from the host cell or from the growthmedium/supernatant. Such methods of production may also comprise a stepof purification of the antibody or protein product and/or formulatingthe antibody or product into a composition including at least oneadditional component, such as a pharmaceutically acceptable carrier orexcipient.

In embodiments when the antibody or protein of the invention is made upof more than one polypeptide chain (e.g., certain fragments such as Fabfragments), then all the polypeptides are preferably expressed in thehost cell, either from the same or a different expression vector, sothat the complete proteins, e.g., binding proteins of the invention, canassemble in the host cell and be isolated or purified therefrom.

The antibodies of the invention may also be used to produce furtherantibodies that bind to CCR4. Such uses involve for example theaddition, deletion, substitution or insertion of one or more amino acidsin the amino acid sequence of a parent antibody to form a new antibody,wherein said parent antibody is one of the antibodies of the inventionas defined elsewhere herein, and testing the resulting new antibody toidentify antibodies that bind to CCR4. Such methods can be used to formmultiple new antibodies that can all be tested for their ability to bindCCR4. Preferably said addition, deletion, substitution or insertion ofone or more amino acids takes place in one or more of the CDR domains.

Such modification or mutation to a parent antibody can be carried out inany appropriate manner using techniques well known and documented in theart, for example by carrying out methods of random or directedmutagenesis. If directed mutagenesis is to be used then one strategy toidentify appropriate residues for mutagenesis utilizes the resolution ofthe crystal structure of the binding protein-antigen complex, e.g., theAb-Ag complex, to identify the key residues involved in the antigenbinding (Davies and Cohen, 1996). Subsequently, those residues can bemutated to enhance the interaction. Alternatively, one or more aminoacid residues can simply be targeted for directed mutagenesis and theeffect on binding to CCR4 assessed.

Random mutagenesis can be carried out in any appropriate way, e.g., byerror-prone PCR, chain shuffling or mutator E. coli strains.

Thus, one or more of the V_(H) domains of the invention can be combinedwith a single V_(L) domain or a repertoire of V_(L) domains from anyappropriate source and the resulting new antibodies tested to identifyantibodies specific for CCR4. Conversely, one or more of the V_(L)domains of the invention can be combined with a single V_(H) domain orrepertoire of V_(H) domains from any appropriate source and theresulting new antibodies tested to identify antibodies that bind toCCR4.

Similarly, one or more, or preferably all three CDRs of the V_(H) and/orV_(L) domains of the invention can be grafted into a single V_(H) and/orV_(L) domain or a repertoire of V_(H) and/or V_(L) domains, asappropriate, and the resulting new antibodies tested to identifyantibodies that bind to CCR4.

The targeted mutations of the CDRs, especially CDR3 of the light and/orheavy chains, have been shown to be an effective technique forincreasing antibody affinity and are preferred. Preferably, blocks of 3to 4 amino acids of the CDR3 or specific regions called “hot-spots” aretargeted for mutagenesis.

“Hot spots” are the sequences where somatic hypermutation takes place invivo (and below Neuberger and Milstein, 1995). The hotspot sequences canbe defined as consensus nucleotide sequences in certain codons. Theconsensus sequence is the tetranucleotide, RGYW, in which R can beeither A or G, Y can be C or T and W can be either A or T (Neuberger andMilstein, 1995). In addition, the serine residues encoded by thenucleotides AGY are predominantly present in the CDRs regions of thevariable domain over those encoded by TCN corresponding to a potentialhot-spot sequences (Wagner et al., 1995).

Thus, the nucleotide sequence of the CDRs of the heavy and light chainsof each antibody of the invention can be scanned for the presence of thehot-spot sequences and AGY codons. The identified hot-spots of the CDRregions of the light and heavy chain can then optionally be compared tothe germinal sequences of the heavy and light chains using theInternational ImMunoGen Tics database (IMGT,http://imgt.cines.fr/textes/vquest/) (Davies et al., 1990). A sequence,identical to the germ line, suggest that somatic mutation has notoccurred; therefore random mutations can be introduced mimicking thesomatic events occurring in vivo or alternatively, site directedmutagenesis can be carried out, e.g., at the hot spots and/or AGYcodons. In contrast, a different sequence shows that some somaticmutations have already occurred. It will remain to be determined if thein vivo somatic mutation was optimal.

Preferred hot-spots for mutation are those that code for exposed aminoacids and preferably those that encode amino acids that form part of theantigen binding sites. Other preferred hot-spots for mutation are thosethat code for non-conserved amino acids. The hot-spots that code forburied or conserved amino acids within the CDRs are preferably notmutagenized. These residues are usually critical for the overallstructure and are unlikely to interact with the antigen since they areburied.

Methods of carrying out the above described manipulation of amino acidsand protein domains are well known to a person skilled in the art. Forexample, said manipulations could conveniently be carried out by geneticengineering at the nucleic acid level wherein nucleic acid moleculesencoding appropriate binding proteins and domains thereof are modifiedsuch that the amino acid sequence of the resulting expressed protein isin turn modified in the appropriate way.

Testing the ability of one or more antibodies to specifically bind toCCR4 can be carried out by any appropriate method, which are well knownand described in the art. CCR4+ cell lines may be obtained from culturecollections, or they may be prepared by transforming CCR4-negative cellswith a construct that allows expression of recombinant CCR4. Such cells,or immobilised CCR4 can readily be used to assay binding, for example byconventional methods such as ELISA, BiaCon, etc.

The new antibodies produced by these methods will preferably haveimproved functional properties, e.g. a higher or enhanced affinity (orat least an equivalent affinity) for CCR4 as the parent antibodies, andcan be treated and used in the same way as the antibodies of theinvention as described elsewhere herein (e.g., for therapy, diagnosis,in compositions etc). Alternatively, or additionally, the new antibodieswill have one or more other improved functional properties as describedelsewhere herein.

New antibodies produced, obtained or obtainable by these methods form ayet further aspect of the invention.

This invention further provides compositions comprising at least onehuman antibody or antibody fragment of the invention, optionallyincluding a diluent. Such compositions may be pharmaceuticallyacceptable compositions or compositions for use in laboratory studies.In terms of the pharmaceutical compositions, they may preferably beformulated for parenteral, intravenous or even subcutaneousadministration.

The present invention provides a number of methods and uses of the humanantibodies and antibody fragments of the invention. Concerning allmethods, the terms “a” and “an” are used to mean “at least one”, “atleast a first”, “one or more” or “a plurality” of steps in the recitedmethods, except where specifically stated. This is particularly relevantto the administration steps in the treatment methods. Thus, not only maydifferent doses be employed with the present invention, but differentnumbers of doses, e.g., injections, may be used, up to and includingmultiple injections. Combined therapeutics may be used, administeredbefore, after or during administration of the anti-CCR4 therapeuticantibody.

Various useful in vitro methods and uses of the antibodies orimmunoconjugates of the invention are provided that have importantbiological implications. First provided are methods of, and uses in,binding CCR4, which generally comprise effectively contacting acomposition comprising CCR4 with at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof. The antibodies ofthe invention, or immunoconjugates thereof, can thus be used in bindingassays. Suitably useful binding assays include those commonly employedin the art, such as in immunoblots, Western blots, dot blots, RIAs,ELISAs, immunohistochemistry, fluorescent activated cell sorting (FACS),immunoprecipitation, affinity chromatography, and the like.

Methods of, and uses in, detecting CCR4 are provided, which generallycomprise contacting a composition suspected of containing CCR4 with atleast a first antibody or immunoconjugate of the invention, orantigen-binding fragment thereof, under conditions effective to allowthe formation of CCR4/antibody complexes and detecting the complexes soformed. The detection methods and uses may be used in connection withbiological samples, e.g., in diagnostics for tumours, and diagnostickits based thereon are also provided.

The methods and uses of the present invention are particularly intendedfor use in animals and patients that have, or are at risk fordeveloping, any disease or condition associated with CCR4 expression oractivity or in which CCR4 plays a biological role. Such diseases anddisorders include diseases which are mediated by CCR4 positive cells,typically CCR4+Th2 or Th17 cells, which, upon binding of a ligand toCCR4, may take part in a signaling pathway which will cause orcontribute to a disorder or disease. They also include diseases causedby aberrant proliferation of cells expressing CCR4. Such aberrantlyproliferating cells may naturally be CCR4+, or they may havemutated/been transformed to express CCR4 As mentioned above, expressionof CCR4 may help cancer cells expressing this antigen to evade theimmune system. Thus, there is provided a method of treating a disease ordisorder mediated by CCR4 and/or characterised by aberrant proliferationof CCR4-positive cells.

Alternatively viewed, there is provided the treatment of a conditionwhich can benefit from one or more of the following

(i) the selective elimination of CCR4+ cells(ii) the inhibition of CCR4 binding to one or more of its ligands(iii) the inhibition of CCR4-mediated cellular responses to a CCR4ligand, particularly the inhibition of chemotaxis or increasedintracellular calcium ion concentration (cell activation).

Preferably, the CCR4 ligand is MDC and/or TARC.

It is well known to those of ordinary skill in the art that as CCR4 isinvolved in a wide range of diseases and disorders, a given anti-CCR4therapy, once shown to be effective in any acceptable model system, canbe used to treat the entire range of diseases and disorders connectedwith CCR4 expression.

In one embodiment, the CCR4-mediated condition is a T-helper cell type2-mediated immune disease. By “T-helper cell type 2-mediated immunedisease” is meant a disease involving immunoglobulin E (IgE) and mastcells due to the development and activation of allergen-specific Th2cells.

The CCR4-mediated disease or disorder may be a disease or conditionassociated with inflammation, infection and/or cancer. Such diseases ordisorders can be treated or prevented with the present antibodies andcompositions. Preferred diseases or conditions include: (1) allergicdiseases such as systemic anaphylaxis or hypersensitivity responses,drug allergies, allergic bronchopulmonary aspergillosis (ABPA), insectsting allergies and food allergies, (2) inflammatory bowel diseases,such as Crohn's disease, ulcerative colitis, ileitis and enteritis, (3)vaginitis, (4) psoriasis and inflammatory dermatoses such as dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria andpruritus, (5) vasculitis, (6) spondyloarthropathies, (7) scleroderma,(8) asthma and respiratory allergic diseases such as allergic asthma,allergic rhinitis, chronic obstructive pulmonary disease,hypersensitivity lung diseases and the like, (9) autoimmune diseases,such as arthritis (including rheumatoid and psoriatic), multiplesclerosis, systemic lupus erythematosus, type I diabetes,glomerulonephritis, and the like, (10) graft rejection (includingallograft rejection and graft-v-host disease), and (11) other diseasesin which undesired inflammatory responses are to be inhibited, such asatherosclerosis, myositis, T-cell mediated neurodegenerative diseases,multiple sclerosis, encephalitis, meningitis, hepatitis, nephritis,sepsis, sarcoidosis, allergic conjunctivitis, otitis, Castleman'sdisease, sinusitis, LPS-induced endotoxic shock, Behcet's syndrome andgout, (12) cancers, both hematological and non-hematological cancers,preferably Breast cancer, Colorectal cancer, Esophageal cancer, Gastriccancer, Hepatocellular carcinoma, Lung cancer, Melanoma, Ovarian cancer,Pancreatic cancer, Adult T-cell leukemia/lymphoma (ATL), PeripheralT-cell lymphoma, unspecified Diffuse large B-cell lymphoma, Hodgkin'slymphoma, B-cell chronic lymphocytic leukemia, CTCL, particularlyMycosis fungoides, Sézary syndrome, cervical cancer, kidney cancer,brain cancer, prostate cancer, stomach cancer (13) infections such asEpstein-Barr virus (EBV) infection, HIV infection and other viralinfections.

The binding of the antibody of the present invention to CCR4 may alsoimpair the ability of CCR4-positive aberrant cells such as cancer cellsto evade the host immune system. The antibodies of the present inventionmay be used to block the suppression of DCs by Treg cells, so there isprovided the use of an anti-CCR4 antibody of the present invention as anadjuvant in a vaccine. The vaccine is preferably a vaccine for cancer oran infectious disease. The vaccine may be a preventative vaccine or acurative vaccine. By “adjuvant” is meant an agent which enhances theimmune response of a host to an antigen. When used as vaccine adjuvants,the antibodies of the present invention are therefore typicallyadministered in conjunction or combined with an antigen against which itis desired to elicit an immune response.

As used herein, the term “aberrant proliferation” means cellproliferation that deviates from the normal, proper, or expected course.For example, aberrant cell proliferation may include inappropriateproliferation of cells whose DNA or other cellular components havebecome damaged or defective.

Aberrant cell proliferation may include cell proliferation whosecharacteristics are associated with an indication caused by, mediatedby, or resulting in inappropriately high levels of cell division,inappropriately low levels of apoptosis, or both. Such indications maybe characterized, for example, by single or multiple local abnormalproliferations of cells, groups of cells, or tissue (s), cancerous ornon-cancerous, benign or malignant.

Any reference to “tumour(s)” herein also refers to “cancer(s)” or“carcinoma(s)”. Metastatic cancers can also be treated, as can thereduction of metastases from a primary tumour. So-called minimalresidual disease (MRD), which is left in post-surgery patients, may beamenable for immunotherapy with anti-CCR4 antibodies.

The present invention thus further provides methods of, and uses in,treating a disease as defined above, comprising administering to ananimal or patient with such a disease, a therapeutically effectiveamount of an anti-CCR4 antibody of the invention, or an antigen-bindingfragment or immunoconjugate of such an anti-CCR4 antibody.

A yet further aspect of the invention provides the use of the antibodiesof the invention or an antigen-binding fragment or immunoconjugate ofsuch an antibody in the manufacture of a composition or medicament foruse in therapy, imaging or diagnosis.

A yet further aspect provides the antibodies of the invention or anantigen-binding fragment or immunoconjugate of such an antibody for usein therapy, diagnosis or imaging.

In addition, the invention provides compositions comprising theantibodies of the invention or an antigen-binding fragment orimmunoconjugate of such an antibody with one or more pharmaceuticallyacceptable excipient, carrier, diluent, buffer or stabilizer.

The in vivo methods as described herein are generally carried out in amammal. Any mammal may be treated, for example humans and any livestock,domestic or laboratory animal. Specific examples include mice, rats,pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however,the mammal is a human.

Thus, the term “animal” or “patient” as used herein includes any mammal,for example humans and any livestock, domestic or laboratory animal.Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits,cows and monkey. Preferably, however, the animal or patient is a humansubject.

This invention links both methods of treating disorders as defined aboveusing unconjugated or naked antibodies and fragments thereof, and CCR4+cell, preferably CCR4+ tumour cell, targeting methods usingimmunoconjugates in which an antibody of the invention orantigen-binding fragment thereof, is operatively attached to atherapeutic agent. Unless otherwise specifically stated or made clear inscientific terms, the terms “antibody and fragment thereof”, as usedherein, therefore mean an “unconjugated or naked” antibody or fragment,which is not attached to another agent, particularly a therapeutic ordiagnostic agent. These definitions do not exclude modifications of theantibody, such as, by way of example only, modifications to improve thebiological half life, affinity, avidity or other properties of theantibody, or combinations of the antibody with other effectors.

The treatment methods and uses of the invention also encompass the useof both unconjugated or naked antibodies and immunoconjugates. In theimmunoconjugate-based treatment methods, an antibody of the invention,or antigen-binding fragment thereof, is preferably operatively attachedto a second therapeutic agent (the anti-CCR4 antibody itself, being thefirst therapeutic agent). The therapeutic agent may for example be ananti-cancer agent or an anti-inflammatory agent, includingcorticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs).

The foregoing treatment methods and uses will generally involve theadministration of the pharmaceutically effective composition to theanimal or patient systemically, such as by transdermal, intramuscular,intravenous injection and the like. However, any route of administrationthat allows the therapeutic agent to localize to the tumour site orsites, will be acceptable. Therefore, other suitable routes of deliveryinclude oral, nasal or respiratory and topical.

“Administration”, as used herein, means provision or delivery ofanti-CCR4 antibody therapeutics in an amount(s) and for a period oftime(s) effective to exert therapeutic, e.g. anti-tumour effects. Thepassive administration of proteinaceous therapeutics is generallypreferred, in part, for its simplicity and reproducibility.

However, the term “administration” is herein used to refer to any andall means by which anti-CCR4 antibodies of the invention are deliveredor otherwise provided to the target site. “Administration” thereforeincludes the provision of cells that produce the anti-CCR4 antibody ofthe invention in a manner effective to result in delivery to the targetsite. In such embodiments, it may be desirable to formulate or packagethe cells in a selectively permeable membrane, structure or implantabledevice, generally one that can be removed to cease therapy. Exogenousanti-CCR4 antibody of the invention will still generally be preferred,as this represents a non-invasive method that allows the dose to beclosely monitored and controlled.

The therapeutic methods and uses of the invention also extend to theprovision of nucleic acids that encode an anti-CCR4 antibody of theinvention in a manner effective to result in their expression in thevicinity of the tumour or their localization to the target site. Anygene therapy technique may be employed, such as naked DNA delivery,recombinant genes and vectors, cell-based delivery, including ex vivomanipulation of patients' cells, and the like.

The anti-CCR4 antibodies of the invention can also be used to deliverother therapeutic or diagnostic agents to the target site In suchembodiments, the other therapeutic or diagnostic agents are generallyoperatively attached to the anti-CCR4 antibodies of the invention.

The “therapeutically effective amounts” for use in the invention areamounts of anti-CCR4 antibody of the invention, or immunoconjugatesthereof, effective to specifically kill at least a portion of targetCCR4+ cells; to specifically induce apoptosis in at least a portion oftarget CCR4+ cells; to specifically induce necrosis in at least aportion of target CCR4+ cells; to inhibit the binding of a CCR4 ligandto CCR4; to inhibit CCR4-mediated cellular responses to a CCR4 ligand,preferably inhibit the increase in intracellular calcium ionconcentration in response to a CCR4 ligand; to reduce inflammation;and/or to induce tumour regression or remission upon administration toanimals or patients having a CCR4+ tumour. Such effects are preferablyachieved while exhibiting little or no binding to, or little or nokilling of cells in normal, healthy tissues; and exerting negligible ormanageable adverse side effects on normal, healthy tissues of the animalor patient.

By “target site” is meant the location of CCR4+ cells which mediate adisorder or which proliferate in an aberrant manner causing orexacerbating a disorder. The target site may thus for example be atumour or the site of CCR4-mediated inflammation. “Target cells” areCCR4+ cells which mediate a disorder or which proliferate in an aberrantmanner causing or exacerbating a disorder. Thus, target cells may forexample include CCR4+ tumour cells, CCR4+ Treg cells and/or CCR4+ Th2cells.

The terms “preferentially” and “specifically”, as used herein in thecontext of killing or inducing apoptosis or of inducing necrosis ofCCR4+ cells such as CCR4+ tumour cells or of reducing inflammation or ofinducing tumour regression or remission, thus mean that the anti-CCR4antibody of the invention or immunoconjugates thereof, function toachieve CCR4+ target cell destruction, e.g. tumour cell destructionand/or tumour necrosis, that is substantially confined to the targetsite, and does not substantially extend to causing destruction and/ortissue necrosis in normal, healthy tissues of the animal or subject.

Anti-CCR4 antibodies of the invention or therapeutic conjugates arepreferably linked to one or more radiotherapeutic agents,chemotherapeutic agents, anti-angiogenic agents, apoptosis-inducingagents, anti-tubulin drugs, anti-cellular or cytotoxic agents, cytokineor chemokine antagonists, inhibitors of cytokine or chemokineexpression, ATPase inhibitors, anti-inflammatory agents, otherantibodies (e.g. as bispecific antibodies) or coagulants (coagulationfactors) or anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs).

The invention thus provides a range of conjugated antibodies andfragments thereof in which the anti-CCR4 antibody is operativelyattached to at least one other therapeutic or diagnostic agent. The term“immunoconjugate” is broadly used to define the operative association ofthe antibody with another effective agent and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. Recombinant fusion proteins areparticularly contemplated. So long as the delivery or targeting agent isable to bind to the target and the therapeutic or diagnostic agent issufficiently functional upon delivery, the mode of attachment will besuitable.

Attachment of agents via the carbohydrate moieties on antibodies is alsocontemplated. Glycosylation, both O-linked and N-linked, naturallyoccurs in antibodies. Recombinant antibodies can be modified to recreateor create additional glycosylation sites if desired, which is simplyachieved by engineering the appropriate amino acid sequences (such asAsn-X-Ser, Asn-X-Thr, Ser, or Thr where X is any amino acid except Pro)into the primary sequence of the antibody.

Currently preferred agents for use in anti-CCR4 antibody or therapeuticconjugates of the invention and related methods and uses are those thatcomplement or enhance the effects of the antibody and/or those selectedfor a particular type of disorder (e.g. tumour type) or patient.

“Therapeutic agents that complement or enhance the effects of theantibody” include radiotherapeutic agents, chemotherapeutic agents,anti-angiogenic agents, apoptosis-inducing agents, anti-tubulin drugs,anti-cellular or cytotoxic agents, coagulants, cytokine or chemokineantagonists, inhibitors of cytokine or chemokine expression, ATPaseinhibitors, anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs),other antibodies, (e.g. as bispecific antibodies), any one or more ofwhich are preferred for use herewith.

Currently preferred anti-cancer, particularly anti-leukaemia agentsinclude Anthracycline drugs such as daunorubicin, Doxorubicin,Cytarabine, 6-thioguanine, Mitoxantrone, busulfan (Myleran®), dasatinib(Sprycel™), prednisone, vincristine sulfate (Oncovin®), Chlorambucil,Fludarabine, Pentostatin and Cladribine.

Currently preferred agents for the treatment of ATL include zidovudine(azidothymidine) and the CHOP regimen. CHOP stands for Cyclophosphamide,Hydroxydaunorubicin (Adriamycin), Oncovin (Vincristine),Prednisone/Prednisolone.

Currently preferred anti-angiogenic agents include angiostatin,endostatin, any one of the angiopoietins, vasculostatin, canstatin andmaspin.

“Anti-tubulin drug(s)”, as used herein, means any agent, drug, prodrugor combination thereof that inhibits cell mitosis, preferably bydirectly or indirectly inhibiting tubulin activities necessary for cellmitosis, preferably tubulin polymerization or depolymerization.Currently preferred anti-tubulin drugs include colchicine, taxol,vinblastine, vincristine, vindescine and one or more of thecombretastatins.

Currently preferred NSAIDs include COX-2 inhibitors, sulphonanilides,licofelone and omega-3 fatty acids

The attachment or association of the preferred agents with anti-CCR4antibodies of the invention gives “immunoconjugates”, wherein suchimmunoconjugates often have enhanced and even synergistic therapeuticproperties, e.g. anti-tumour or anti-inflammatory properties.

The use of anti-cellular and cytotoxic agents results in anti-CCR4antibody “immunotoxins” of the invention, whereas the use of coagulationfactors results in anti-CCR4 antibody “coaguligands” of the invention.

The use of at least two therapeutic agents is also contemplated, such ascombinations of one or more radiotherapeutic agents, chemotherapeuticagents, anti-angiogenic agents, apoptosis-inducing agents, anti-tubulindrugs, anti-cellular and cytotoxic agents, cytokine or chemokineantagonists, inhibitors of cytokine or chemokine expression, ATPaseinhibitors, anti-inflammatory agents such as corticosteroids, preferablyglucocorticoids, or non-steroidal anti-inflammatory drugs (NSAIDs),other antibodies, (e.g. as bispecific antibodies) and coagulationfactors.

In certain applications, the anti-CCR4 antibody therapeutics of theinvention will be operatively attached to cytotoxic, cytostatic orotherwise anti-cellular agents that have the ability to kill or suppressthe growth or cell division of cells. Suitable anti-cellular agentsinclude chemotherapeutic agents, as well as cytotoxins and cytostaticagents. Cytostatic agents are generally those that disturb the naturalcell cycle of a target cell, preferably so that the cell is taken out ofthe cell cycle.

Exemplary chemotherapeutic agents include: hormones, such as steroids;anti-metabolites, such as cytosine arabinoside, fluorouracil,methotrexate or aminopterin; anthracyclines; mitomycin C; vincaalkaloids; antibiotics; demecolcine; etoposide; mithramycin; andanti-tumor alkylating agents, such as chlorambucil or melphalan. Certainpreferred anti-cellular agents are DNA synthesis inhibitors, such asdaunorubicin, doxorubicin/adriamycin, and the like. Overall,taxol/paclitaxel, docetaxel, cisplatin, gemcitabine, a combretastatinand doxorubicin/adriamycin are currently preferred anti-cancer agents.

V-type ATPase inhibitors are also currently preferred, such assalicylihalamide, concanamycin or bafilomycin, as are protein synthesisinhibitors, such as psymberin, pederin, irciniastatin A.

In certain therapeutic applications, toxin moieties will be preferred,due to the much greater ability of most toxins to deliver a cell killingeffect, as compared to other potential agents. Therefore, certainpreferred anti-cellular agents for anti-CCR4 antibody constructs of theinvention are plant-, fungus- or bacteria-derived toxins. Exemplarytoxins include epipodophyllotoxins; bacterial endotoxin or the lipid Amoiety of bacterial endotoxin; ribosome inactivating proteins, such assaporin or gelonin; a-sarcin; aspergillin; restrictocin; ribonucleases,such as placental ribonuclease; diphtheria toxin and pseudomonasexotoxin. Currently preferred examples are ricin, gelonin, abrin,diphtheria, pseudomonas and pertussis toxins.

Certain preferred toxins are the A chain toxins, such as ricin A chain.The most preferred toxin moiety is often ricin A chain that has beentreated to modify or remove carbohydrate residues, so called“deglycosylated A chain” (dgA). Deglycosylated ricin A chain ispreferred because of its extreme potency, longer half-life, and becauseit is economically feasible to manufacture it a clinical grade andscale. Recombinant and/or truncated ricin A chain may also be used.

The anti-CCR4 antibody therapeutics of the invention may comprise acomponent that is capable of promoting coagulation, i.e., a coagulant.Here, the targeting antibody may be directly or indirectly, e.g., viaanother antibody, linked to a factor that directly or indirectlystimulates coagulation.

Preferred coagulation factors for such uses are Tissue Factor (TF) andTF derivatives, such as truncated TF (tTF), dimeric, trimeric,polymeric/multimeric TF, and mutant TF deficient in the ability toactivate Factor VII. Other suitable coagulation factors include vitaminK-dependent coagulants, such as Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa; vitamin K-dependent coagulation factors thatlack the Gla modification; Russell's viper venom Factor X activator;platelet-activating compounds, such as thromboxane A₂ and thromboxane A₂synthase; and inhibitors of fibrinolysis, such as α2-antiplasmin.Overall, truncated Tissue Factor (tTF) is currently preferred.

The preparation of immunoconjugates and immunotoxins is generally wellknown in the art (see, e.g., U.S. Pat. No. 4,340,535). Each of thefollowing patents are further incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingimmunotoxin generation, purification and use: U.S. Pat. Nos. 6,004,554;5,855,866; 5,965,132; 5,776,427; 5,863,538; 5,660,827 and 6,051,230.

A variety of chemotherapeutic and other pharmacological agents can alsobe successfully conjugated to anti-CCR4 antibody therapeutics of theinvention. Exemplary antineoplastic agents that have been conjugated toantibodies include doxorubicin, daunomycin, methotrexate andvinblastine. Moreover, the attachment of other agents such asneocarzinostatin, macromycin, trenimon and α-amanitin has been described(see U.S. Pat. Nos. 5,660,827; 5,855,866; and 5,965,132; eachincorporated herein.)

The preparation of coaguligands is also easily practiced. The operableassociation of one or more coagulation factors with an anti-CCR4antibody of the invention may be a direct linkage, such as thosedescribed above for the immunotoxins. Alternatively, the operativeassociation may be an indirect attachment, such as where the antibody isoperatively attached to a second binding region, preferably an antibodyor antigen binding region of an antibody, which binds to the coagulationfactor. The coagulation factor should be attached to the anti-CCR4antibody of the invention at a site distinct from its functionalcoagulating site, particularly where a covalent linkage is used to jointhe molecules.

Bispecific or trispecific antibodies may also be employed in the methodsof the invention. In such antibodies one arm binds to CCR4 and is anantibody of the present invention. Methods for preparing bispecificantibodies are well known and described in the art.

In the preparation of immunoconjugates, immunotoxins and coaguligands,recombinant expression may be employed. The nucleic acid sequencesencoding the chosen anti-CCR4 antibody of the invention, and therapeuticagent, toxin or coagulant, are attached in-frame in an expressionvector. Recombinant expression thus results in translation of thenucleic acid to yield the desired immunoconjugate. Chemicalcross-linkers and avidin:biotin bridges may also join the therapeuticagents to the anti-CCR4 antibody of the invention.

The compositions and methods of the present invention may be used incombination with other therapeutics and diagnostics. In terms ofbiological agents, preferably diagnostic or therapeutic agents, for use“in combination” with an anti-CCR4 antibody in accordance with thepresent invention, the term “in combination” is succinctly used to covera range of embodiments. The “in combination” terminology, unlessotherwise specifically stated or made clear from the scientificterminology, thus applies to various formats of combined compositions,pharmaceuticals, cocktails, kits, methods, and first and second medicaluses.

The “combined” embodiments of the invention thus include, for example,where the anti-CCR4 of the invention is a naked antibody and is used incombination with an agent or therapeutic agent that is not operativelyattached thereto. In such cases, the agent or therapeutic agent may beused in a non-targeted or targeted form. In “non-targeted form”, theagent, particularly therapeutic agents, will generally be used accordingto their standard use in the art. In “targeted form”, the agent willgenerally be operatively attached to a distinct antibody or targetingregion that delivers the agent or therapeutic agent to the targetdisease site. The use of such targeted forms of biological agents, bothdiagnostics and therapeutics, is also quite standard in the art.

In other “combined” embodiments of the invention, the anti-CCR4 antibodyof the invention is an immunoconjugate wherein the antibody is itselfoperatively associated or combined with the agent or therapeutic agent.The operative attachment includes all forms of direct and indirectattachment as described herein and known in the art.

The “combined” uses, particularly in terms of an anti-CCR4 antibody ofthe invention in combination with therapeutic agents, also includecombined compositions, pharmaceuticals, cocktails, kits, methods, andfirst and second medical uses wherein the therapeutic agent is in theform of a prodrug. In such embodiments, the activating component able toconvert the prodrug to the functional form of the drug may again beoperatively associated with the anti-CCR4 antibodies of the presentinvention.

In certain preferred embodiments, the therapeutic compositions,combinations, pharmaceuticals, cocktails, kits, methods, and first andsecond medical uses will be “prodrug combinations”. As will beunderstood by those of ordinary skill in the art, the term “prodrugcombination”, unless otherwise stated, means that the antibody of theinvention is operatively attached to a component capable of convertingthe prodrug to the active drug, not that the antibody is attached to theprodrug itself. However, there is no requirement that the prodrugembodiments of the invention need to be used as prodrug combinations.Accordingly, prodrugs may be used in any manner that they are used inthe art, including in ADEPT and other forms.

Thus, where combined compositions, pharmaceuticals, cocktails, kits,methods, and first and second medical uses are described, preferably interms of diagnostic agents, and more preferably therapeutic agents, thecombinations include anti-CCR4 antibodies that are naked antibodies andimmunoconjugates, and wherein practice of the in vivo embodiments of theinvention involves the prior, simultaneous or subsequent administrationof the naked antibodies or immunoconjugate and the biological,diagnostic or therapeutic agent; so long as, in some conjugated orunconjugated form, the overall provision of some form of the antibodyand some form of the biological, diagnostic or therapeutic agent isachieved.

The foregoing and other explanations of the effects of the presentinvention on tumors are made for simplicity to explain the combined modeof operation, type of attached agent(s) and such like. This descriptiveapproach should not be interpreted as either an understatement or anoversimplification of the beneficial properties of the anti-CCR4antibodies of the invention. It will therefore be understood that suchantibodies themselves have anti-CCR4 properties and thatimmunoconjugates of such antibodies will maintain these properties andcombine them with the properties of the attached agent; and further,that the combined effect of the antibody and any attached agent willtypically be enhanced and/or magnified.

The invention therefore provides compositions, pharmaceuticalcompositions, therapeutic kits and medicinal cocktails comprising,optionally in at least a first composition or container, a biologicallyeffective amount of at least a first anti-CCR4 antibody of theinvention, or an antigen-binding fragment or immunoconjugate of such ananti-CCR4 antibody; and a biologically effective amount of at least asecond biological agent, component or system.

The “at least a second biological agent, component or system” will oftenbe a therapeutic or diagnostic agent, component or system, but it neednot be. For example, the at least a second biological agent, componentor system may comprise components for modification of the antibodyand/or for attaching other agents to the antibody. Certain preferredsecond biological agents, components or systems are prodrugs orcomponents for making and using prodrugs, including components formaking the prodrug itself and components for adapting the antibodies ofthe invention to function in such prodrug or ADEPT embodiments.

Where therapeutic or diagnostic agents are included as the at least asecond biological agent, component or system, such therapeutics and/ordiagnostics will typically be those for use in connection with thetreatment or diagnosis of one or more of the disorders defined above.

Thus, in certain embodiments “at least a second therapeutic agent” willbe included in the therapeutic kit or cocktail. The term “at least asecond therapeutic agent” is chosen in reference to the anti-CCR4antibody of the invention being the first therapeutic agent. Theantibodies of the invention may thus be combined with chemotherapeuticagents, radiotherapeutic agents, cytokine or chemokine antagonists,inhibitors of cytokine or chemokine expression, anti-angiogenic agents,apoptosis-inducing agents or anti-cancer immunotoxins or coaguligands,or anti-inflammatory agents including corticosteroids and NSAIDs, someexamples of which are discussed elsewhere herein.

Other exemplary anti-cancer agent include, e.g., neomycin,podophyllotoxin(s), TNF-α, α_(v)β₃ antagonists, calcium ionophores,calcium-flux inducing agents, and any derivative or prodrug thereof.Currently preferred anti-tubulin drugs include colchicine, taxol,vinblastine, vincristine, vindescine, a combretastatin or a derivativeor prodrug thereof.

In terms of compositions, kits and/or medicaments of the invention, thecombined effective amounts of the therapeutic agents may be comprisedwithin a single container or container means, or comprised withindistinct containers or container means. The cocktails will generally beadmixed together for combined use. Agents formulated for intravenousadministration will often be preferred. Imaging components may also beincluded. The kits may also comprise instructions for using the at leasta first antibody and the one or more other biological agents included.

Speaking generally, the at least a second therapeutic agent may beadministered to the animal or patient substantially simultaneously withthe anti-CCR4 antibody of the invention; such as from a singlepharmaceutical composition or from two pharmaceutical compositionsadministered closely together.

Alternatively, the at least a second therapeutic agent may beadministered to the animal or patient at a time sequential to theadministration of the anti-CCR4 antibody of the invention. “At a timesequential”, as used herein, means “staggered”, such that the at least asecond anti-cancer agent is administered to the animal or patient at atime distinct to the administration of the anti-CCR4 antibody of theinvention. Generally, the two agents are administered at timeseffectively spaced apart to allow the two agents to exert theirrespective therapeutic effects, i.e., they are administered at“biologically effective time intervals”. The at least a secondtherapeutic agent may be administered to the animal or patient at abiologically effective time prior to the anti-CCR4 antibody of theinvention, or at a biologically effective time subsequent to thattherapeutic.

Accordingly, the present invention provides methods for treating ananimal or patient with a tumor, comprising:

(a) subjecting the animal or patient to a first treatment thatsubstantially reduces the tumor burden; and(b) subsequently administering at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof; optionally whereinthe antibody or fragment is operatively associated with a secondtherapeutic agent.

Preferred first treatments include surgical resection andchemotherapeutic intervention.

In other embodiments, the present invention provides methods fortreating an animal or patient with a CCR4-mediated disorder, comprising:

(a) subjecting the animal or patient to a first treatment thatsubstantially reduces the CCR4-mediated burden such as inflammation; and(b) subsequently administering at least a first anti-CCR4 antibody ofthe invention, or antigen-binding fragment thereof; optionally whereinthe antibody or fragment is operatively associated with a secondtherapeutic agent.

In certain other embodiments, the antibodies and immunoconjugates of theinvention may be combined with one or more diagnostic agents, typicallydiagnostic agents for use in connection with the diagnosis of a disorderas defined above. A range of diagnostic compositions, kits and methodsare thus included within the invention.

Yet further aspects are methods of diagnosis or imaging of a subjectcomprising the administration of an appropriate amount of an antibody orother protein of the invention as defined herein to the subject anddetecting the presence and/or amount and/or the location of the antibodyor other protein of the invention in the subject.

In one embodiment, the invention provides a method of reducingimmunosuppression associated with CCR4 expression in an animal,comprising administering to said animal the antibody of the invention,or an immunoconjugate thereof, in an amount effective to form complexesbetween said antibody and CCR4 in said animal, thereby reducingimmunosuppression associated with CCR4 expression in an animal.

Appropriate diseases to be imaged or diagnosed in accordance with theabove described uses and methods include any disease and preferably anycancer as described elsewhere herein.

In one embodiment, the invention provides a method of diagnosing diseaseor monitoring the progress of disease in an animal comprising the stepof:

(a) contacting a test sample taken from said animal with an antibody ofthe invention or an immunoconjugate thereof.

In a further embodiment, the invention provides a method of diagnosingdisease or monitoring the progress of disease in an animal comprisingthe steps of:

(a) contacting a test sample taken from said animal with an antibody ofthe invention or an immunoconjugate thereof;(b) measuring or detecting the presence and/or amount and/or location ofantibody-antigen complex in the test sample; and, optionally(c) comparing the presence and/or amount of antibody-antigen complex inthe test sample to a control.

In the above methods, said contacting step is carried out underconditions that permit the formation of an antibody-antigen complex.Appropriate conditions can readily be determined by a person skilled inthe art.

In the above methods any appropriate test sample may be used, forexample biopsy cells, tissues or organs suspected of being affected bydisease or histological sections.

In certain of the above methods, the presence of any amount ofantibody-antigen complex in the test sample would be indicative of thepresence of disease. Preferably, for a positive diagnosis to be made,the amount of antibody-antigen complex in the test sample is greaterthan, preferably significantly greater than, the amount found in anappropriate control sample. More preferably, the significantly greaterlevels are statistically significant, preferably with a probabilityvalue of <0.05. Appropriate methods of determining statisticalsignificance are well known and documented in the art and any of thesemay be used.

Monitoring the progress of a disease may also involve monitoring thepresence and/or amount of antibody-antigen complex in test samples overtime. Thus, monitoring may involve (d) comparing the presence and/oramount of antibody-antigen complex in a first test sample to thepresence and/or amount of antibody-antigen complex in a second testsample taken from said animal. By “first test sample” is meant a samplethat was taken prior to taking the “second test sample”, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, weeks, months or years priorto taking the second test sample. A decrease in the amount ofantibody-antigen complex in the second test sample compared to the firsttest sample is indicative of the disease regressing, whereas an increaseis indicative of the disease progressing.

Appropriate control samples could be readily chosen by a person skilledin the art, for example, in the case of diagnosis of a particulardisease, an appropriate control would be a sample from a subject thatdid not have that disease. Appropriate control “values” could also bereadily determined without running a control “sample” in every test,e.g., by reference to the range for normal subjects known in the art.

For use in the diagnostic or imaging applications, the antibodies of theinvention may be labeled with a detectable marker such as a radio-opaqueor radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; aradioactive emitter (e.g., α, β or γ emitters); a fluorescent(fluorophore) or chemiluminescent (chromophore) compound, such asfluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such asalkaline phosphatase, beta-galactosidase or horseradish peroxidase; animaging agent; or a metal ion; or a chemical moiety such as biotin whichmay be detected by binding to a specific cognate detectable moiety,e.g., labelled avidin/streptavidin. Methods of attaching a label to abinding protein, such as an antibody or antibody fragment, are known inthe art. Such detectable markers allow the presence, amount or locationof binding protein-antigen complexes in the test sample to be examined.

Preferred detectable markers for in vivo use include an X-ray detectablecompound, such as bismuth (III), gold (III), lanthanum (III) or lead(II); a radioactive ion, such as copper⁶⁷, gallium⁶⁷, gallium⁶⁸,indium¹¹¹, indium¹¹³, iodine¹²³, iodine¹²⁵, iodine¹³¹, mercuryl⁹⁷,mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³,technetium^(99m) or yttrium⁹⁰; a nuclear magnetic spin-resonanceisotope, such as cobalt (II), copper (II), chromium (III), dysprosium(III), erbium (III), gadolinium (III), holmium (Ill), iron (II), iron(III), manganese (II), neodymium (III), nickel (II), samarium (III),terbium (III), vanadium (II) or ytterbium (Ill); or rhodamine orfluorescein.

The invention also includes diagnostic or imaging agents comprising theantibodies of the invention attached to a label that produces adetectable signal, directly or indirectly. Appropriate labels aredescribed elsewhere herein.

The invention further includes kits comprising one or more of theantibodies, immunoconjugates or compositions of the invention or one ormore of the nucleic acid molecules encoding the antibodies of theinvention, or one or more recombinant expression vectors comprising thenucleic acid sequences of the invention, or one or more host cells orviruses comprising the recombinant expression vectors or nucleic acidsequences of the invention. Preferably said kits are for use in themethods and uses as described herein, e.g., the therapeutic, diagnosticor imaging methods as described herein, or are for use in the in vitroassays or methods as described herein. The antibody in such kits maypreferably be an antibody conjugate as described elsewhere herein, e.g.,may be conjugated to a detectable moiety or may be an immumoconjugate.Preferably said kits comprise instructions for use of the kitcomponents, for example in diagnosis. Preferably said kits are fordiagnosing or treating diseases as described elsewhere herein andoptionally comprise instructions for use of the kit components todiagnose or treat such diseases.

The antibodies of the invention as defined herein may also be used asmolecular tools for in vitro or in vivo applications and assays. As theantibodies have an antigen binding site, these can function as membersof specific binding pairs and these molecules can be used in any assaywhere the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent thatcomprises an antibody of the invention as defined herein and the use ofsuch antibodies as molecular tools, for example in in vitro or in vivoassays.

Cancer treatment may also be carried out by:

(a) forming an image of a tumor by administering to an animal or patienthaving a tumor a diagnostic amount of at least a firstdetectably-labeled anti-CCR4 antibody of the invention, comprising adiagnostic agent operatively attached to the anti-CCR4 antibody of theinvention, thereby forming a detectable image of the tumor; and(b) subsequently administering to the same animal or patient atherapeutically optimized amount of at least a first naked anti-CCR4antibody of the invention or therapeutic agent-antibody construct usingsuch an antibody, thereby causing an anti-tumor effect.

The invention will now be described in more detail in the followingnon-limited examples with reference to the Tables and Figures in which:

Table 1 lists some of the sequences disclosed herein relating toantibody 17G

Table 2 lists some of the sequences disclosed herein relating toantibody 9E

Table 3 lists some of the sequences disclosed herein relating toantibody 1O

Table 4 lists some of the sequences disclosed herein relating toantibody 11F

Table 5 lists some of the sequences disclosed herein relating to the IgGform of antibody 17 G. The variable regions are underlined.

Table 6 lists some of the sequences disclosed herein relating to IgGform of antibody 9E. The variable regions are underlined.

Table 7 lists some of the sequences disclosed herein relating to IgGform of antibody 1O. The variable regions are underlined.

Table 8 lists some of the sequences disclosed herein relating to IgGform of antibody 11F. The variable regions are underlined.

Table 9 shows the calculated affinities (K_(D) values) from IgGtitrations on CCR4⁺ cells using “One site—Specific binding” model of thesoftware Prism (GraphPad, San Diego, Calif.). The IgG forms ofantibodies 17G, 9E and KM3060var using various CCR4⁺ cell types wereused (see Example 2).

Table 10 shows the sequences of antibody KM3060var.

Table 11 lists some of the sequences disclosed herein relating toantibody 9E10J.

Table 12 lists some of the sequences disclosed herein relating toantibody 9E1D.

Table 13 lists some of the sequences disclosed herein relating to IgGform of antibody 9E10J. The variable regions are underlined.

Table 14 lists some of the sequences disclosed herein relating to IgGform of antibody 9E1 D. The variable regions are underlined.

Table 15 shows apparent affinities (IC₅₀ and K_(D)) determined fromresults obtained in Example 4.

Table 16 shows IC₅₀ values determined in Ca⁺⁺ flux assays (Example 5).

Table 17 shows a comparison of ADCC activity of anti-CCR4 antibodies(Example 6).

Table 18 shows a comparison of ADCC activity of anti-CCR4 antibodiesincluding defucosylated 9E10J (Example 11).

Table 19 shows some of the results of Example 14. Apparent affinities(K_(D)) as determined by saturated antibody titration on cells in flowcytometry.

Table 20 shows some of the results of Example 9. IC₅₀ values andcalculated affinities (K_(D)) for cell-binding inhibition ofbiotinylated MDC, as determined from competition binding experiments onDT-40-CCR4⁺ and CCRF-CEM cells.

Table 21 shows some of the results of Example 9. IC₅₀ values forcell-binding inhibition of biotinylated TARC, as determined fromcompetition binding experiments on DT-40-CCR4⁺ and CCRF-CEM cells.

Table 22 shows an overview of determined IC₅₀-values from competitionexperiments of Example 9.

Table 23 shows CDR consensus sequences based on the antibodies disclosesherein.

Table 24 shows some results of Example 15 concerning the determinationof the effect of 9E10J on platelet aggregation. Platelets were isolatedfrom fresh donor-blood and incubated in presence of either 9E10J-IgG (10μg/ml) or native ligands MDC and TARC (0.25 μg/ml). ADP (3 μM) was usedas control to demonstrate that platelets behave as expected. Anti-GFP(10 μg/ml) was included into the measurements as well to demonstratespecificity of the assay. Aggregation was determined and expressed in %by measuring the absorbance in the sample chambers which were comparedto a platelet-depleted plasma (=0% aggregation) in parallel.Preincubation=IgG-samples were first incubated with the platelets for 10min, followed by addition of the ligands; mixture=IgG-samples were mixedwith the ligands and followed by incubation with the platelets.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 17G. ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 2 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 9E. ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 3 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 1O. ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 4 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 11F ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 5 illustrates some of the results of the flow cytometry of Example2. The CCR4-specificity of the antibodies 17G IgG and 9E IgG wasconfirmed by staining CCR4 transfected and untransfected DT40 cells. Theanti-CCR4 antibody KM3060var IgG served as a positive control andanti-GFP IgG was used as a negative control in this experiment

FIG. 6 illustrates some of the results of the flow cytometry of Example2. The binding of anti-CCR4 antibodies KM3060var, 17G and 9E to CCR4expressing cell line CCRF-CEM was analyzed using flow cytometry.Staining of the cells was performed in triplicate and error-barsindicate the standard deviations which are too small to be seen. Thenegative control antibody anti-GFP did not give any staining signals.

FIG. 7 illustrates some of the results of the flow cytometry of Example2. The CCR4-specificity of the 1O antibody was confirmed by stainingCCR4 transfected and untransfected DT40-cells. The anti-GFP scFv servedas a negative control in this experiment.

FIG. 8 illustrates some of the results of the flow cytometry of Example2. The CCR4-specificity of the 11F antibody was confirmed by stainingCCR4 transfected and untransfected DT40-cells. The anti-VZV scFv servedas a negative control in this experiment.

FIG. 9 is a graph which shows some of the results of Example 3. Theanti-CCR4 antibodies 11F, 9E, 17G and 1O interfere with the ligand MDCbinding to CCR4 transfected DT40 cells as determined by flow cytometryby staining cells with the scFvs after pre-incubation with differentconcentrations of MDC. In accordance with previous reports, the antibodyKM3060var did not interfere the MDC binding to the CCR4 receptor.

FIGS. 10 A, B and C are graphs showing the results of the competitionassay of Example 4. The binding of antibody to CCR4 is shown as thepercentage of maximum binding (i.e. binding in the absence of anycompetitor) plotted against increasing concentrations of the testantibodies (i.e. the putative competitors).

FIG. 10 A shows binding of 0.28 nM biotinylated 17G IgG to CCR4transfected DT40-cells in presence of non-biotinylated 17G, 9E andKM3060var IgGs. KM3060var does not have any appreciable effect on thebinding of 17G to CCR4, whereas the presence of antibody 9E causes areduction in the amount of 17G that binds to CCR4, indicating that 17Gand 9E compete with one another for binding to CCR4.

FIG. 10B shows binding of 7 nM biotinylated 9E IgG to CCR4 transfectedDT40-cells in presence of non-biotinylated 17G, 9E and KM3060var.KM3060var does not have any appreciable effect on the binding of 9E toCCR4, whereas the presence of antibody 17G causes a reduction in theamount of 9E that binds to CCR4, confirming the results of FIG. 10A that17G and 9E compete with one another for binding to CCR4.

FIG. 10C shows binding of 6.8 nM biotinylated KM3060var IgG to CCR4transfected DT40-cells in presence of non-biotinylated 17G, 9E andKM3060var. It can be seen that neither 17G nor 9E compete with KM3060varfor binding to CCR4.

FIG. 11 shows the results of Example 5 in which calcium flux inhibitionby 9E, 17G and KM3060var IgG was assayed. The natural CCR4+CCRF-CEM cellline, labeled with Fluo-4, was preincubated with antibodies followed bystimulation with TARC (A) or MDC (B) ligands. The recording of the cellsstarted about 15 sec after the ligand addition. TARC and MDC both causecalcium flux, seen as a high peak in the Figures. TARC-induced calciumflux is almost completely inhibited by 17G and 9E respectively, but notby KM3060var. MDC-induced calcium flux is also strongly inhibited by 17Gand 9E respectively, but not by KM3060var.

FIG. 12 shows antibody-dependent cell cytotoxicity (ADCC) results ofExample 6. The figure shows that 17G (B) and 9E (A) are able to induceADCC of CCRF-CEM cells in the presence of human PBMCs.

FIG. 13 is a graph which shows some of the results from Example 2. TheCCR4-specificity of the antibodies 9E10J and 9E1 D was confirmed bystaining CCR4 transfected and untransfected DT40 cells. 9E served as apositive control in this experiment. Binding to CCR4+ cells is shownusing light grey bars. Binding to CCR4− cells is shown using dark greybars, the absence of a dark grey bar meaning that no significant bindingcould be detected.

FIG. 14 is a graph which shows some of the results from Example 3.Binding of anti-CCR4 scFv antibodies 17G, 9E, 1O, 11F and KM3060var toCCR4-transfected DT-40 cells was assayed in the presence or absence ofthe CCR4 ligand TARC, as determined by flow cytometry.

FIG. 15 shows some of the results from Example 4. Competition titrationof fixed amounts of biotinylated IgG1 9E at concentration of either 0.7(a, c) or 3.3 nM (b) in presence of increasing concentrations ofunlabelled IgGs 9E, 9E10J or KM3060var on DT-40-CCR4⁺ (a-b) or CCRF-CEMcells (c). Detection of bound biotinylated IgG was performed byStrep-PE. MFI=median fluorescence intensity

FIG. 16 shows some of the results from Example 5. Ca⁺⁺-flux assays werecarried out on CCRF-CEM cells labelled with Fluo-4 using overlappingconcentration intervals. Cells were pre-incubated with 9E, 9E10J, orKM3060var IgGs for 15 min before adding the CCR4-specific ligand TARC.The IgG concentrations were 0.001 μg/ml (a), 0.01 μg/ml (b), 0.1 μg/ml(c), 0.1 μg/ml (e), 1.0 μg/ml (f) and 10 μg/ml (g). The areas under thecurves (AUC) were integrated using software Prism (GraphPad) and plottedas percentage of AUC for maximal stimulation with TARC alone, as shownin panels (d) and (h) for the left and right columns, respectively.

FIG. 17 shows some of the results from Example 5. Ca⁺⁺-flux assays werecarried out on CCRF-CEM cells labelled with Fluo-4 using overlappingconcentration intervals. Cells were pre-incubated with 9E10J orKM3060var IgGs for 15 min before adding the CCR4-specific ligand TARC. Abroad range of IgG concentrations was used. Signals were recorded andthe areas under the curves (AUC) were integrated using software Prism(GraphPad) and plotted as percentage of AUC for maximal stimulation withTARC alone.

FIG. 18 shows some of the results from Example 6, concerning ADCC ofCCRF-CEM cells mediated by anti-CCR4 antibodies. FIG. 18 (a) comparisonof IgG 9E to IgG 9E10J, FIG. 18 (b) comparison of 9E10J to KM3060var.

FIG. 19 shows some of the results from Example 10. Binding ofdefucosylated 9E10J (9E10Jdef), unmodified 9E10J and KM3060var to DT40cells, stably transfected with CCR4 (+) or untransfected, i.e.CCR4-negative (−). Presented is the binding signal at a concentration of20 μg/ml. Binding was detected using anti-human-FITC (fluoresceinisothiocyanate) (goat) conjugated-IgG.

FIG. 20 shows some of the results from Example 11. ADCC of CCRF-CEMcells mediated by anti-CCR4 antibodies, comparing defucosylated 9E10J(9E10Jdef) to unmodified 9E10J and KM3060var.

FIG. 21 shows some of the results from Example 14. Binding titration ofIgGs 9E, 9E1 D and KM3060var on DT-40 CCR4⁺ cells (a) and on humanT-cell leukaemia cells CCRF-CEM (b). Detection was performed withanti-human-FITC. MFI, median fluorescence intensity

FIG. 22 shows some of the results from Example 14. Binding titration ofscFv 9E, 9E1 D and 9E10J on DT-40 CCR4⁺ cells. The scFvs werecross-linked via myc-tag in order to simulate a dimeric IgG-alikesituation. Detection was performed with anti-human-PE. MFI, medianfluorescence intensity

FIG. 23 shows some of the results from Example 14. Binding titration ofIgGs 9E, 9E10J and KM3060var on human T-cell leukaemia cells CCRF-CEM.Detection was performed with anti-human-PE. MFI, median fluorescenceintensity

FIG. 24 shows some of the results from Example 9. Competition titrationof fixed amounts of biotinylated MDC (ca. 10 nM in a, b and ca. 20 nM inc, d) in presence of increasing concentrations of either unlabelledantibody (scFv or IgG 9E10J) or CCR4 ligand (MDC) on DT-40-CCR4⁺ (a) orCCRF-CEM cells (b-d). Detection of bound biotinylated MDC was performedby Strep-PE. MFI, median fluorescence intensity.

FIG. 25 shows some of the results from Example 9. Competition titrationof fixed amounts of biotinylated TARC (ca. 20 nM) in presence ofincreasing concentrations of either unlabelled antibody (IgG 9E10J) orCCR4 ligands, MDC or TARC on DT-40-CCR4⁺ (a,b) or CCRF-CEM cells (c,d).Detection of bound biotinylated TARC was performed using Strep-PE. In(b) and (d), incubation of proteins with cells was performed in presenceof 1% BSA. MFI, median fluorescence intensity

FIG. 26 shows some of the results from Example 9. Competition titrationof fixed amounts of biotinylated MDC (a) and TARC (b) in presence ofincreasing amounts of unlabelled antibodies 9E, 9E10J or unlabelledligands.

FIG. 27 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 9E10J. ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 28 shows the nucleotide and amino acid sequence of inter alia theheavy and light chain of clone 9E1 D. ScFv was cloned via NcoI/NotI siteinto pHOG21. The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic. The c-myc epitope and 6 His areunderlined and double underlined, respectively.

FIG. 29 shows some results of Example 13. Inhibition of ligand (TARC;3.5 nM) induced cell migration by anti-CCR4 antibodies and controls.Human T-cell leukaemia CCRF-CEM cells were induced to migrate inMultiscreen-MIC chambers, where the ligand TARC was placed in the lowerchamber and the cells were co-incubated with antibodies or medium onlyas a control in the upper chambers. Migrated cells to the lower chamberwere detected and counted. Mean and SD values of triplicates areplotted. (a) Inhibition of TARC-induced migration of 9E10J in comparisonto 9E and anti-GFP. (b) Inhibition of TARC-induced migration of 9E10J incomparison to 1 G1 and KM3060var.

FIG. 30 shows some results from Example 16. Staining of peripheric bloodlymphocytes (PBL). Lymphoctyes were gated out of the total cellpopulation. T-cells were gated upon incubation with anti-CD3-APC-IgGs(human) and anti-CD8-PE-Cy5-IgGs (human). Biotinylated 9E10J andbiotinylated comparator KM3060var were titrated from 20 μg/ml down to0.3 ng/ml and detected with PE-conjugated Streptavidin. were used PBLswere a.) Gating of lymphocyte population. b.) Gating of CD3-positivecells and sub-dividing into CD8-positive (CD4-negative) and CD8-negative(CD4-positive) cells. c.) Titration curves of 9E10J and KM3060var ongated CD8-negative (CD4-positive) cells, expressed in % of positiveevents.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Novel Antibodies

Four human antibodies have been identified which can specifically bindto CCR4. Single chain forms of the antibodies were cloned in the pHOG21plasmid which contains a c-myc and 6×His tag epitopes. TG1 bacteria weretransformed, and the scFv was expressed upon IPTG induction. The bindingof the purified scFv was confirmed by EasyCyte.

The nucleotide sequences of the heavy and light chain of the antibodyproducing clones were sequenced. The antibodies are designated as 17G,9E, 11F, 1O, 9E10J and 9E1 D. The nucleotide sequence and amino acidsequence of the light and heavy chain of 17G are shown in FIG. 1. TheCDR regions of the light and heavy chains of 17G are shown in Table 1.The nucleotide sequence and amino acid sequence of the light and heavychain of 9E are shown in FIG. 2. The CDR regions of the light and heavychains of 9E are shown in Table 2. The nucleotide sequence and aminoacid sequence of the light and heavy chain of 1O are shown in FIG. 3.The CDR regions of the light and heavy chains of 1O are shown in Table3. The nucleotide sequence and amino acid sequence of the light andheavy chain of 11F are shown in FIG. 4. The CDR regions of the light andheavy chains of 11F are shown in Table 4.

Two further antibodies have been identified which can specifically bindto CCR4. Single chain forms of the antibodies were prepared as describedabove. The nucleotide sequence and amino acid sequence of the light andheavy chain of 9E10J are shown in FIG. 27. The CDR regions of the lightand heavy chains of 9E10J are shown in Table 11. The nucleotide sequenceand amino acid sequence of the light and heavy chain of 9E1 D are shownin FIG. 28. The CDR regions of the light and heavy chains of 9E1 D areshown in Table 12.

The IgG form of antibodies 17G, 9E, 1O, 11F, 9E10J and 9E1 D has alsobeen made. IgGs were prepared using standard protocols. Briefly, thegenes encoding the corresponding variable domains were cloned into themammalian expression vector pLNO comprising the genes for human constantdomains (Norderhaug et al, 1997). The antibodies were expressed in acell factory, and the first harvest was purified on a protein A columnand fractionated into monomer by size exclusion chromatography. The IgGsretained their ability to specifically bind to CCR4.

The IgG form is of the IgG1 isotype and it comprises two heavy chainsand two light chains. Each heavy chain comprises a VH domain of SEQ IDNO: 69 (for 17G), SEQ ID NO: 71 (for 9E or 9E1 D), SEQ ID NO:73 (for1O), SEQ ID NO:75 (for 11F), or SEQ ID NO: 105 (for 9E10J), and a humanIgG1 constant region. Each light chain comprises a VL domain of SEQ IDNO: 70 (for 17G), SEQ ID NO: 72 (for 9E or 9E10J), SEQ ID NO: 74 (for1O) SEQ ID NO: 76 (for 11F), or SEQ ID NO: 115 (for 9E1 D), and a humanlambda light constant region, except for the IgG form of 11F, which hasa human kappa light chain. The full IgG sequences of 17G, 9E, 1O, 11F,9E10J and 9E1 D are shown in Tables 5, 6, 7, 8, 13 and 14 respectively.

A further antibody, 9E1 Dv, has been identified. This antibody has apoint mutation at position 88 in FR3 (numbering according to Kabat etal., Sequences of proteins of immunological interest; Fifth edition,1991), where 9E1Dv has an Alanine (A) instead of the Valine (V) found in9E1 D. The properties of 9E1 Dv, insofar as they have been tested,appear to correspond to those of antibody 9E1 D.

Example 2 Binding of Anti-CCR4 Antibodies to Target Expressing Cells

To demonstrate the CCR4-specificity of the antibodies disclosed inExample 1 and to estimate their binding affinities, in-house CCR4transfected and untransfected HEK293T-cells, DT40-cells and the naturalCCR4⁺ CCRF-CEM cell line were used in flow cytometry for staining withIgGs 17G and 9E as well as with scFvs 1O and 11F. As a positive control,in-house cloned and expressed KM3060var IgG which is specific for anepitope present in positions 2-29 in N-terminal part of human CCR4(EP1270595) was used. An anti-GFP antibody (raised against the greenfluorescent protein) or the anti-VZV antibody (raised against Varicellazoster virus) were used as negative controls (anti-GFP both as scFv andIgG, while anti-VZV only as scFv).

CCRF-CEM (acute lymphoblastic leukemia, ATCC number CCL-119), HEK293T/17(human kidney, ATCC number CRL-11268), and DT40 (chicken lymphoma, ATCCnumber CRL-2111) cell lines were obtained from the American Type CultureCollection (ATCC, Rockville, Md.). The CCRF-CEM and DT40 cells weremaintained in RPMI-1640 culture medium and the HEK293T cells weremaintained in Dulbecco's Modified Eagle Medium (DMEM) culture medium.All cells were maintained with fetal calf serum, the concentration was10% for DT40 and HEK293T cells and 20% for CCRF-CEM cells. All mediawere supplemented with Penicillin and Streptomycin.

For the flow cytometry experiments, the cells were harvested from theculture flasks, washed 2 times with PBS, re-suspended in PBS with 0.2%BSA and 0.09% NaN₃ and finally aliquoted 1×10⁵ cells per well intoV-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany). Cellswere centrifuged at 400×g for 5 min and then incubated at 4° C. for 45min with different antibody dilutions.

For staining with scFvs, the scFv preparations were pre-incubated with achimeric (mouse variable/human constant domains) anti-c-myc antibodyprior adding to the cells.

After washing with PBS with 0.2% BSA and 0.09% NaN₃, the cells werestained with 10 μg/ml of RPE-conjugated goat anti-human IgG (AbDSerotec,Düsseldorf, Germany) for 30 minutes at 4° C. The stained cells werewashed, re-suspended in 200 μl PBS with 0.2% BSA and 0.09% NaN₃ andtransferred to a U-shaped 96-well plate (Corning, Schiphol-Rijk, TheNetherlands) for acquisition on EasyCyte flow cytometer (GuavaTechnologies, Hayward, Calif., USA).

The results obtained clearly indicate that the antibodies 17G, 9E, 1Oand 11F are specific for CCR4. Binding of antibodies 17G and 9E toCCR4-positive DT40 cells compared to CCR4-negative DT40 cells isillustrated in FIG. 5. Binding of 17G and 9E to CCR4-expressing CCRF-CEMcells is illustrated in FIG. 6. Selective binding of 17G and 9E to CCR+HEK293T cells compared to CCR-HEK293T cells was also found.

The affinity values (K_(D)) deduced from these IgG titration experimentswere estimated to be in low nanomolar range (see Table 9).

The specificity of antibody 1O scFv for CCR4 is illustrated in FIG. 7and the specificity of antibody 11F scFv for CCR4 is illustrated in FIG.8.

The binding specificity of 9E10J and 9E1 D was also tested againstuntransfected and CCR4-transfected DT40 cells under conditionscorresponding essentially to those described above. For this experiment,9E10J and 9E1 D were expressed as scFvs having a c-myc tag at the 3′end, which was used to create dimers to mimic the IgG format. Theantibodies were pre-incubated with an anti-c-myc antibody prior toadding to cells. Staining was carried out essentially as describe above.The results show that 9E10J and 9E1 D bind selectively to CCR4-positivecells (FIG. 13).

Example 3 Anti-CCR4 Antibodies Interference with Ligand Binding

To determine whether the anti-CCR4 antibodies interfere with the bindingof CCR4 ligands to the receptor, MDC (obtained from PeproTech EC Ltd.,London, UK) was pre-incubated with CCR4-transfected DT40-cells prior tostaining with the scFvs. As a negative control, the KM3060var scFv (seeTable 10 for sequence) was used which is specific for the N-terminalpart of human CCR4. It was shown previously that the antibody KM3060does not interfere with ligand binding to the receptor (T Ishida et al(2006) Specific Recruitment of CC Chemokine Receptor 4—PositiveRegulatory T Cells in Hodgkin Lymphoma Fosters Immune Privilege, CancerRes 66: (11): 5716-5722.). KM3060var corresponds to KM3060, but it hasbeen expressed in a different host cell, which may have led todifferences in the sugar chains attached to this antibody.

The CCR4+ DT40-cells were harvested from culture flasks, washed 2 timeswith RPMI-1640 culture medium and aliquoted 1×105 cells per well intoV-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany). Cellswere centrifuged at 500×g for 5 minutes and then incubated for 30minutes at 37° C. with 0, 1 or 10 ng/ml of MDC (PeproTech EC, London,UK) in RPMI-1640 culture medium. The supernatants were aspirated after acentrifugation step at 500×g for 5 minutes and cells were incubated for1 hour at 4° C. with 0.5 μg/ml of scFv and 2.5 μg/ml of an in-houseproduced anti-cMyc with human Fc. After washing three times with PBScontaining 0.2% BSA and 0.09% NaN3, the cells were stained with 2.5μg/ml of RPE-conjugated goat anti-human IgG (AbDSerotec, Düsseldorf,Germany) for 1 hour at 4° C. Cells were washed, re-suspended in 200 μlPBS with 0.2% BSA and 0.09% NaN3 and transferred to a U-shaped 96-wellplate (Corning, Schiphol-Rijk, The Netherlands) for flow cytometry usingan EasyCyte device (Guava Technologies, Hayward, Calif., USA). Theresults showed a clear decrease in staining signals, apart from stainingwith KM3060var antibody, after pre-incubation of CCR4 transfected DT40cells with 10 ng/ml of MDC (FIG. 9). This indicates that the antibodies17G, 9E, 1O and 11F interfere with the ligand binding and thus have aCCR4-blocking activity.

A similar experiment showed that all four antibodies 17G, 9E, 1O and 11Falso interfere with the binding of TARC to CCR4. The results are shownin FIG. 14.

Example 4 Competition Between Anti-CCR4 Antibodies

To analyze the epitope binding of anti-CCR4 antibodies 17G, 9E andKM3060var, competition experiments were performed using flow cytometry.The binding of biotinylated antibodies to CCR4 transfected DT40-cellswas challenged in the presence of different concentrations ofnon-biotinylated anti-CCR4 antibodies. The CCR4⁺ DT40 cells wereharvested from culture flasks, washed 2 times with PBS, re-suspended inPBS with 0.2% BSA and 0.09% NaN₃ and finally aliquoted 1×10⁵ cells perwell into V-shaped 96-well plates (Greiner Bio-One, Frickenhausen,Germany). Cells were centrifuged at 400×g for 5 min and then incubatedat 4° C. for 3-4 hours with fixed amounts of biotinylated IgG anddifferent concentrations of non-biotinylated antibodies. After washingwith PBS with 0.2% BSA and 0.09% NaN₃, the cells were stained with 2.5μg/ml of Streptavidin—RPE (BD Pharmingen, San Diego, Calif., USA) for 30minutes at 4° C. The stained cells were washed, re-suspended in 250 μlPBS with 0.2% BSA and 0.09% NaN₃ and transferred to a U-shaped 96-wellplate (Corning, Schiphol-Rijk, The Netherlands) for acquisition onEasyCyte flow cytometer (Guava Technologies, Hayward, Calif., USA). Theresults shown in FIGS. 10 A, B and C illustrate that the 17G and 9Eantibodies have epitopes that are identical or at least overlapping.There was no competition between KM3060var antibody binding to the cellsand the other two antibodies indicating that 17G and 9E do not bind tothe same epitope as KM3060var.

Further competition experiments were carried out to assess whether ornot the test antibodies recognise the same or overlapping epitopes, andto determine their relative affinities for CCR4. Antibodies 9E10J, 9Eand KM3060var were tested using labelled 9E as a competitor.

DT40 and CCRF-CEM cells were cultivated as described in Example 2. Thecells were harvested from culture flasks, washed 2 times with PBS(400×g, 5 min, 4° C.) and resuspended in PBS containing 0.2% BSA and0.09% NaN3. 1×105 cells per well were aliquoted into V-shaped 96-wellplates (Griner Bio-One, Frikenhausen, Germany). Cells were centrifuged(400×g, 5 min, 4° C.) and resuspended in 50 μl IgG 9E10J or KM3060var(dilution series starting at 5 μg/ml) mixed with biotinylated competitorantibody 9E (0.1 or 0.5 μg/ml). The cells were incubated for 2 hours at4° C., mixing with a pipette every 45 min. The cells were washed threetimes with 150 μl PBS with 0.2% BSA and 0.09% NaN3 and stained with 3μg/ml streptavidin PE (BD Biosciences) for 45 minutes at 4° C. Cellswere washed, resuspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3, andtransferred to a U-shaped 96 well Costar (Corning, Schiphol-Rijik, TheNetherlands) plate for flow cytometry using an FACS Canto II flowcytometer (BD Biosciences, Heidelberg, Germany).

The results shown in FIG. 15 demonstrate that both 9E and 9E10Jeffectively inhibited cell binding of biotinylated IgG 9E in adose-dependent manner. In contrast, nearly no decrease of bindingsignals was observed when KM3060var was titrated with biotinylated 9E,thus confirming that epitopes recognized by these antibodies do notoverlap.

To determine IC50 values (antibody concentrations leading to 50%inhibition of biotinylated IgG binding), the experimental data werefitted by non-linear regression curve fit using a model “log (inhibitor)vs. response” of software Prism (GraphPad). Apparent binding affinities(K_(D)) were calculated according to the following equation derived frompreviously described methods (Kipriyanov et al, 1997b; Schodin & Kranz,1993):

K _(D) ^((I)) =IC ₅₀/(1+[bio-IgG^((9E)) ]/K _(D) ^((9E))),

where I is the unlabelled inhibitor (IgG), [bio-IgG^((9E))] is fixedconcentration of biotinylated IgG 9E, and K_(D) ^((9E)) is the bindingaffinity of IgG 9E derived from saturation titration experiments (4.95nM). Results are shown in Table 15.

Example 5 Antagonistic Activity

The ability of 9E and 17G to reduce ligand induced calcium flux in thenatural CCR4⁺ CCRF-CEM cell line was assessed. The antibody KM3060varwas used as a negative control. The sequence-identical defucosylatedclone KM2760 has been reported to be not inhibiting CCR4 signaling. TheCCRF-CEM target cells, cultivated under regular conditions, weresedimented by centrifugation and resuspended twice in RPMI-1640 culturemedium. One ml with 2.5×10⁶ cells was mixed with Fluo-4-AM (Invitrogen,Carlsbad, Calif.), Pluronic F-127 (Invitrogen, Carlsbad, Calif.) andProbenecid to final concentrations of 1 μM, 0.02% and 1 mM respectively.The cells were incubated at 37° C. for 30 min on a vertical rotatingwheel (7 rpm). All subsequent steps were done in the presence of 1 mMProbenecid. The cells were washed twice in RPMI-1640 with 10% FCS, oncein assay buffer (145 mM NaCl, 4 mM KCl, 1 mM NaH₂PO₄, 0.8 mM MgCl₂, 25mM Hepes, 22 mM glucose) and then resuspended to a final density of1.2×10⁶ cells/ml. Equal volumes of cells, assay buffer with or withoutantibodies and ligand (MDC or TARC) were mixed. The first two components(cells and antibodies) were pre-incubated for 15 min prior to additionof the ligand. The final concentrations of the IgGs, TARC and MDC were10 μg/ml, 10 ng/ml and 2.5 ng/ml, respectively. The samples wereimmediately analyzed using the 515-545 nm band pass filter on aFACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany).

The results shown in FIG. 11 clearly demonstrated that both 9E and 17Gact as inhibitors of TARC and MDC. Cells preincubated with 9E, 17G andKM3060var IgGs at concentration 10 μg/mL showed signal responses to TARCstimulation of 3%, −1% and 104% assuming the signal to TARC alone as100% (FIG. 11A). Accordingly, the cells preincubated with 9E, 17G andKM3060var IgGs showed signal responses to MDC stimulation of 64%, 42%and 92%, respectively of the signal to MDC in the absence of IgGs (FIG.11B). No signal was detected when either 9E or 17G IgGs were added tothe cells instead of TARC or MDC ligands (data not shown). The 9E or 17GIgGs also did not show any inhibition of the CXCR4 specific SDF-1 ligand(data not shown).

The experiment was also carried out under similar conditions usingantibody 9E10J in the human IgG1 format, except that incubation of thecells with Fluo4-AM was carried out for 40 min at 37° C. withoutrotating wheel. Antibody KM3060var was included as a negative controland antibody 9E was also included (both also in human IgG1 format). Theresults shown in FIG. 16 demonstrate dose-dependent inhibition ofTARC-induced signalling by both 9E and 9E10J antibodies.

Detailed analysis of Ca⁺⁺ flux inhibition was performed using the broadrange of IgG concentrations for 9E10J (FIG. 17). IC₅₀ (ng/ml) valueswere calculated as explained in Example 4. Complete signal inhibitionwas reached at concentrations 100-300 ng/ml (0.7-2.0 nM) with thecalculated IC₅₀ value of 43.3 pM (Table 16).

Example 6 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The ability of 9E and 17G to induce ADCC was assessed on the naturalCCR4⁺ CCRF-CEM cell line. The CCRF-CEM target cells, cultivated underregular conditions, were sedimented by centrifugation and resuspended inRPMI-1640 culture medium. This step was repeated once. One ml with2.5×10⁶ cells was mixed with calcein-AM (Invitrogen, Carlsbad, Calif.)to a final concentration of 10 μM and then incubated at 37° C. for 30min on a vertical rotating wheel (7 rpm). The cells were washed threetimes in RPMI-1640 with 10% FCS and the cell density was adjusted to3×10⁵/ml. Separately, peripheral blood mononuclear cells (PBMC) wereprepared following conventional procedures (enriched by FicoII—Hypaquegradient centrifugation), washed in RPMI-1640 with 10% FCS andresuspended at 6×10⁶ per milliliter. Fifty μl of each target andeffector cells were added to the same wells in a 96-well microtiterplate giving a ratio of effector to target cells (E:T) of 20:1. Theantibodies (all in IgG format) were added to the same wells in a volumeof 20 μl giving a final concentration range of 10 to 10 000 ng/mL, andwith each concentration in quadruplicate samples. The microtiter platewas then incubated four hours at 37° C., and 20 μl 0.9% Triton-X100 wasadded to some of the wells after 3 hrs and 45 minutes to achievecomplete lysis of the target cells. One hundred μl supernatant of eachsample was then transferred to a black microtiter plate and thefluorescence (excitation: 488 nm, emission: 518 nm) was analyzed in aTECAN M200 plate reader. The fluorescence intensity in the samples withno antibodies was subtracted from the intensity of all other samples.The percentage of lysis in samples with antibodies was estimated basedon fluorescence intensity of the samples with 100% cell lysis aftertreatment with TritonX-100. The dose-response curves were computed bynonlinear regression analysis and a three-parameter fit model usingsoftware Prism (GraphPad, San Diego, Calif., USA).

The results shown in FIGS. 12A and 12B clearly demonstrated that both 9Eand 17G were able to induce ADCC in the presence of human PBMCs withEC₅₀ values of 619 ng/ml and 46 ng/ml and maximum killing of 55% and 22%respectively.

The ability of antibody 9E10J to induce ADCC of CCRF-CEM cells was alsotested and compared to antibody 9E and antibody KM3060var underconditions similar to those described above, except that a rotatingwheel was not used. The results, shown in FIG. 18 and Table 17, showthat 9E and 9E10J can induce ADCC and that ADCC-induction by 9E10J issuperior to ADCC induction by KM3060var.

Example 7 Preparation of Anti-cMyc Hybrid/Human IgG1 Antibody

Anti-cMyc hybrid/human IgG1 antibody: 9E10 H-chain (AJ000488) andlight-chain 9E10 L-chain (AJ000489) variable DNA sequences were orderedfrom GeneArt and converted into a hybrid Human IgG1/Kappa protein.Cloning of the variable VH and VL regions by PCR with specific primerson the scFv template, digested and ligated into heavy- and light-chainvectors, respectively according to the method of Norderhaug et al, JIM204:77-87. HEK293/T cells were transiently transfected and after 5-6days, the IgG was purified via Protein-A followed by size exclusion toisolate the monomeric IgG fraction.

Example 8 Species Cross-Reactivity

Antibodies 9E and 9E10J were tested for their ability to cross-reactwith CCR4 from species other than human.

Hek293T/17 were cultured in DMEM (from PAA, cat# E15-810) with 10% FBS(from PAA cat# A15-252) and 1× penicillin/streptomycin (from PAAcat#P11-010). They were split 2-3× per week. Hek293T/17 were seeded to32000 cells/cm2 for 48 h and 27000 cells/cm2 over the week end.

For transient transfection, Hek293T/17 cells were seeded as 2×106 cellsin a T75 (NUNC) flask. 48 h after seeding, the cells were transfectedwith Fugene (ROCHE). 40 μl Fugene and 16 μg DNA are used per T75. Thecells were used for assays 48 h after transfection.

HEK-293T/17 cells, were transiently transfected either with pcDNA3.1plasmid encoding human CCR4 or with pLNO plasmids (Norderhaug et al,1997) encoding either mouse (GeneBank CAA62372) or monkey (Macacamulatta) CCR4 (PubMed access number XP_001098807) using Fugene (Roche)as a transfection reagent. Non-transfected (CCR4 negative) cells servedas a negative control. The cells were cultivated further for 48 hoursunder regular conditions described above. The cells were washed twicewith PBS, detached from the culture flasks by incubating with Accutase(PAA laboratories, Linz, Austria) for 3 minutes at room temperature. Thecells were then washed 2 times with PBS (400×g, 5 min, 4° C.) andresuspended in PBS containing 0.2% BSA and 0.09% NaN₃. 1×10⁵ cells perwell were aliquoted into V-shaped 96-well plates (Griner Bio-One,Frikenhausen, Germany). Cells were centrifuged (400×g, 5 min, 4° C.) andresuspended in 50 μl of 10 μg/ml 9E or 9E10J, in PBS containing 0.2% BSAand 0.09% NaN3. The cells were incubated further at 4° C. for 60 min.The samples were then washed twice by centrifugation and re-suspensionin 100 μl FACS buffer. The cell pellets were finally re-suspended in 50μl with 3 μg/ml anti-human-IgG-PE (BD Biosciences) and incubated at 4°C. for 45 min. The samples were washed twice as described above andre-suspended in 250 μl FACS buffer followed by transfer into a U-shaped96-well plate (Corning, Schiphol-Rijk, Netherlands) for flow cytometryon FACSCantoll (BD Biosciences, San Jose, Calif.).

9E and 9E10J both strongly bound to human and monkey CCR4; no binding tomurine CCR4 was detected.

Example 9 Inhibition of Ligand Binding

Antibodies 9E and 9E10J were tested for their ability to inhibit bindingof MDC and TARC to CCR4+ cells in a competition experiment usingbiotinylated ligands. The experiment is designed to give an indicationof the affinity of the antibodies for CCR4 compared to affinity of theligands for CCR4.

The competition binding assays were performed with biotinylated MDC orbiotinylated TARC on DT-40-CCR4+ and CCRF-CEM cells. DT40 and CCRF-CEMcells were cultured as described in Example 2. TARC and MDC werebiotinylated using standard procedures. The biotinylation process causessome losses, so the concentrations of biotinylated MDC and TARC recitedherein are approximate.

The cells were harvested from culture flasks, washed 2 times with PBS(400×g, 5 min, 4° C.) and resuspended in PBS containing 0.2% BSA and0.09% NaN3. 1×105 cells per well were aliquoted into V-shaped 96-wellplates (Greiner Bio-One, Frikenhausen, Germany). Cells were centrifuged(400×g, 5 min, 4° C.) and resuspended in 50 μl IgG, single chain orunlabeled ligand (1:10 dilution series starting on 100/500/26, 4 μg/ml)mixed with a fixed concentration of biotinylated ligand (10 or 20 nM,see legend to FIG. 24). The cells were incubated for 2 hours at 4° C.and mixed with a pipette every 45 min. The cells were washed three timeswith 150 μl PBS with 0.2% BSA and 0.09% NaN3 and stained with 3 μg/mlstreptavidin PE (BD Biosciences) for 45 minutes at 4° C. Cells werewashed, resuspended in 200 μl PBS with 0.2% BSA and 0.09% NaN3, andtransferred to a U-shaped 96 well Costar (Corning, Schiphol-Rijik, TheNetherlands) plate for flow cytometry using an FACS Canto II flowcytometer (BD Biosciences, Heidelberg, Germany). To determine IC50values (concentration of inhibitor leading to 50% decrease of signal),the experimental data were fitted by non-linear regression curve fitusing a model “log (inhibitor) vs. response” of software Prism(GraphPad). Apparent binding affinities (KD) were calculated accordingto the following equation derived from previously described methods(Kipriyanov et al, 1997b; Schodin & Kranz, 1993):

KD(I)=IC50/(1+[bio-MDC]/KD(MDC)),

where I is the unlabelled inhibitor (antibody or ligand), [bio-MDC] isfixed concentration of biotinylated MDC, and KD(MDC) is the bindingaffinity of MDC, 0.18 nM (Imai et al, 1998).

The results of competition binding experiments shown in FIG. 24 andTable 20 demonstrate that CCR4-binding of high-affinity ligand MDC caneffectively be inhibited in a dose-dependent manner by IgG 9E10J.CCR4-transfected DT-40 cells express a much higher density of CCR4 thanCCRF-CEM cells which have 15-fold fewer CCR4 receptors on their surface.

It should be noted that unlabelled MDC was able to completely inhibitbinding of biotinylated MDC to CCRF-CEM cells while antibody 9E10J wasnot (FIG. 24). This observation indicates possible binding of MDC toanother receptor on CCRF-CEM cells. Most probably, this is an atypicalscavenger (decoy) GPCR receptor D6 which is able to bind MDC with highaffinity (Graham, 2009; Locati et al, 2005). This also explains thehigher apparent IC50.

Similar binding inhibition experiments were performed using biotinylatedTARC. As expected, TARC, antibody 9E10J and MDC all competed effectivelywith labelled TARC (FIG. 25). To avoid non-specific cell-binding of thelabelled ligand, half of the experiments were performed in the presenceof 1% BSA as a blocking agent. The presence or absence of BSA did notseem to have any significant effect (BSA present in FIGS. 25 b and d,absent in FIGS. 25 a and c).

Example 10 Generation of Defucosylated 9E10J

It has been reported that ADCC enhancement can be achieved bymanipulating the state of oligosaccharides on human IgG1 subclass (NiwaR et al, CanRes, Vol. 64, 2004). Modifying the amount of fucose wasdemonstrated to have a beneficial effect on enhancement of ADCC.Therefore, antibody 9E10J was produced in the presence of Kifunensine, aselective inhibitor of class I α-mannosidases, causing a stop infucosylation of the IgG during production in cell culture. For qualitycontrol, the defucosylated 9E10J IgG was first tested for bindingspecificity on DT40 cells, stably transfected with CCR4 and compared tounmodified 9E10J and KM3060var.

IgGs were produced in the presence of Kifunensine (100 ng/ml; Sigma).After harvesting the cell medium, IgGs were isolated first upon affinitypurification using a ProteinA column (HiTrap, 5 ml, ProteinA; GE). IgGswere eluted using citrate buffer (pH 3) and transferred into 1MTris-buffer (pH 9). Prior a second purification, IgG samples wereup-concentrated and loaded on to a size exclusion chromatography (HiLoadSephadex 200, GE; running buffer PBS pH 7.4). Monomeric fractions werecollected and IgGs were up-concentrated a second time. For bindingexperiments, IgGs were serial diluted 2-fold, starting at aconcentration from 20 μg/ml down to 0.15625 μg/ml. IgGs were incubatedon DT40 cells (1.5*106/ml), stably transfected with human CCR4.Untransfected DT-40 cells were used as a negative control. Bound IgGswere detected via anti-human-FITC (goat) conjugated antibodies.

The results presented in the FIG. 19 demonstrate that the modificationof the fucosylation pattern has no influence on the CCR4 bindingcapabilities of 9E10J.

Example 11 Induction of Antibody Dependent Cellular Cytotoxicity (ADCC)of Defucosylated IgG1-Variant

Defucosylated 9E10J prepared as described in Example 10 was compared tounmodified 9E10J and unmodified KM3060var. The IgGs were tested usingCCRF-CEM cells which constitutively express CCR4. ADCC induction wasassayed as described in Example 6.

The results shown in FIG. 20 and Table 18 demonstrate that thedefucosyalted variant of candidate 9E10J has increased killingproperties of CCR4-positive CCRF-CEM cells by human PBMCs and possessesmuch higher cytolytic activity in comparison to the unmodified versionof 9E10J (4 to 5-fold lower EC50)

Example 12 Inhibition of Growth and Induction of Apoptosis

The ability of antibody 9E to induce apoptosis in Ramos cells (aB-lymphoblastoid cell line derived from a Burkitt lymphoma). was assayedusing Annexin V staining and FACS analysis. No significant induction ofapoptosis was observed.

A growth inhibition test was carried out using 9E, 9E10J and KM3060var(all in IgG format) on CCRF-CEM cells and Ramos cells (aB-lymphoblastoid cell line derived from a Burkitt lymphoma). No growthinhibition was observed.

Example 13 Inhibition of Chemotaxis

i) The inhibition of chemotaxis by antibodies 9E and 9E10J was assayedby contacting CCR4+cells capable of chemotaxis with antibody 9E or 9E10Jin one chamber, and a ligand of CCR4 (MDC or TARC) was placed in anotherchamber separated from the first chamber by a membrane or filter havinga suitable pore size. The effect of the antibody on cell migrationtowards the ligand (chemotaxis) was determined by comparing chemotaxisin the presence of the antibody to chemotaxis in the absence of theantibody. Both 9E and 9E10J were found to inhibit chemotaxis ofCCR4-positive cells towards MDC and TARC.

ii) The ability of 9E10J to reduce ligand induced migration of thenatural CCR4+CCRF-CEM cells was assessed and compared with 9E, KM3060varand 1G1 antibodies. All antibodies were tested in IgG1 format.

CCRF-CEM target cells, cultivated as described in example 2, weresedimented by centrifugation and re-suspended twice in RPMI-1640 culturemedium without FCS. In a third centrifugation step, cells werere-suspended in RPMI-1640 culture medium supplemented with 1% FCS andadjusted to a final density of 1.6×107 cells/ml. In parallel, 150 μl ofRPMI supplemented with 1% FCS containing TARC at 3.5 nM was added intoeach of the lower chamber of a Multiscreen-MIC plate 96 well (8 μmpores, Millipore, Billerica, Mass., USA, MAMIC5S10). 50 μl of the cellswith IgGs (serial diluted from 167 nM down to 0.3 nM) were added to theupper compartment of the Multiscreen-MIC 96 chamber plate and incubatedat 37° C. for 3 hr. The filter was removed and 100 μl of cells (from thelower chamber) were transferred after re-suspending into a 96-wellCOASTAR plate (Greiner Bio-One, Frikenhausen, Germany); an additionalvolume of 100 μl of PBS (supplemented with 0.2% BSA and 0.09% NaN3) wasadded to the samples. Migration was evaluated by counting of gatedcells.

The data presented in FIG. 29 demonstrate inhibition of TARC inducedchemotaxis of CCRF-CEM cells by 9E and 9E10J. No significant inhibitionof chemotaxis was observed for a antibodies KM3060var and 1 G1 or forthe control IgG (anti-GFP).

Example 14 Binding Specificity and Affinity for CCR4

The quality of the specific binding to DT40+ cells was tested for 9E1 Dand 9E10J in scFv or IgG format in a titration experiment.

DT 40 and CCRF-CEM cells were cultured as described in Example 2. TheDT40 CCR4+ and DT40 (CCR4−) cells were harvested from culture flasks,washed 2 times with PBS (400×g, 5 min, 4° C.) and resuspended in PBScontaining 0.2% BSA and 0.09% NaN₃. 1×10⁵ cells per well were aliquotedinto V-shaped 96-well plates (Greiner Bio-One, Frikenhausen, Germany).The cells were centrifuged (400×g, 5 min, 4° C.) and resuspended in 50μl ScFvs (5 μg/ml) in PBS containing 0.2% BSA and 0.09% NaN₃, and anin-house produced anti-cMyc antibody with a human Fc (25 μg/ml). After 1hour incubation (4° C.), the cells were washed three times with 150 μlPBS with 0.2% BSA and 0.09% NaN₃ and stained with 2.5 μg/ml RPEconjugated goat anti-human IgG (AbDSerotec, Düsseldorf, Germany) for 45minutes at 4° C. Cells were washed, resuspended in 200 μl PBS with 0.2%BSA and 0.09% NaN₃, and transferred to a U-shaped 96 well Costar(Corning, Schiphol-Rijik, The Netherlands) plate for flow cytometryusing an Easy Cyte device (Guava Technologies, Hayward, Calif., USA).

Alternatively, 9E1D and 9E10J were converted into the full-length humanIgG1 and tested for cell binding. The DT40+CCR4 and DT40 cells wereharvested from culture flasks, washed 2 times with PBS (400×g, 5 min, 4°C.) and resuspended in PBS containing 0.2% BSA and 0.09% NaN₃. 1×10⁵cells per well were aliquoted into V-shaped 96-well plates (GreinerBio-One, Frikenhausen, Germany). Cells were centrifuged (400×g, 5 min,4° C.) and resuspended in a 1:5 dilution series of IgG starting on 10μg/ml (50 μl/well). After 1 hour incubation at 4° C., the cells werewashed three times with 150 μl PBS with 0.2% BSA and 0.09% NaN₃ andstained with 2.5 μg/ml RPE conjugated goat anti-human IgG (AbDSerotec,Düsseldorf, Germany) or 2.5 μg/ml anti human IgG Fc (goat polyclonalaff. pur) FITC (Sigma Aldrich) for 45 minutes at 4° C. Cells werewashed, resuspended in 200 μl PBS with 0.2% BSA and 0.09% NaN₃, andtransferred to a U-shaped 96 well Costar (Corning, Schiphol-Rijik, TheNetherlands) plate for flow cytometry using an Easy Cyte device (GuavaTechnologies, Hayward, Calif., USA) or FACS Canto II flow cytometer (BDBiosciences, Heidelberg, Germany).

The apparent affinity values (K_(D)) were deduced from non-linear fit ofthe experimental data curves using “One site-Specific binding” model ofsoftware Prism (GraphPad, San Diego, Calif.). KM3060var was included aspositive control, as it is known to bind to CCR4. Results are shown inFIGS. 21, 22 and 23 and in Table 19.

Example 15 Aggregation Measurements on Platelets

CCR4 is known to be expressed on platelets, and its ligands MDC and TARCare known to induce platelet aggregation. This could potentially beproblematic, as IgG molecules have two ligand binding sites, so there isa possibility that an IgG capable of recognizing CCR4 may be able tocross link platelets if both arms are able to bind to CCR4 on differentplatelets. This might result in blot clotting in vivo. Therefore, theeffect of 9E10J-IgG on platelet aggregation was examined. 9E10J-IgG wasincubated with isolated platelets alone or in combination with theligands MDC or TARC. ADP, a well-described inducer of aggregation (Varonand Spectre “Antiplatelet agents” Hematology Am Soc Hematol EducProgram. 267-72, 2009), was included as positive control during eachmeasurement.

A total of 30 ml of fresh donor blood was collected into sodium citratecontaining tubes. Platelet rich plasma was obtained by centrifuging atroom temperature (RT) for 15 min at 185 g. The platelet containingplasma was transferred into a fresh tube. To define the baseline,platelet depleted plasma was prepared by centrifugation of 1 ml of theplatelet rich plasma for 5 min at 1200 g at RT, the supernatanttransferred into a new tube and treated in the following the same way asthe platelet rich sample. Aggregation measurements are performed at 37°C. under stirring using a Packs-4 aggregometer (Helena Biosciences,USA). IgGs (9E10J and anti-GFP as negative control) were incubated withplatelets at a concentration of 10 μg/ml in FACS buffer (PBS, pH 7.4,0.2% BSA, 0.09% NaN₃). ADP served as a positive control for aggregationand was used at a final concentration of 3 μM. Ligands (MDC and TARC)were tested 0.25 μg/ml.

The signal obtained with ADP was set as 100%, the baseline 0 was definedby the platelet depleted serum, which was measured in parallel in allexperimental settings

To check that the tested IgGs bind to the isolated platelets, FACSstaining of platelets was performed in parallel. Platelets weretransferred into V-shaped 96 well plates (Greiner Bio-One, Frikenhausen,Germany) and washed once by adding 100 μl of FACS-buffer (1×PBS, pH 7.4,supplemented with 0.02% BSA and 0.09% NaN₃). Platelets were thenincubated for 1 h at 4° C. in presence of 9E10J-IgG (at 10 μg/ml) andwashed afterwards twice in presence of 100 μl of FACS-buffer (1×PBS, pH7.4, supplemented with 0.02% BSA and 0.09% NaN₃). Bound IgG was detectedusing anti-human-PE (1:400 dilutions) after incubation on theIgG-stained cells for 1 h at 4° C. Stained platelets were analyzed inFACS measurements.

Binding of 9E10J-IgG to platelets was observed (data not shown). Theresults are of the aggregation measurements are summarized in Table 24.It is concluded that although 9E10J-IgG binds to platelets, it has noeffect on platelet aggregation: it does not induce aggregation byitself, nor does it inhibit ligand-induced platelet aggregation.

Example 16 Binding of IgG 9E10J to Peripheral Blood Lymphocytes (PBL)

As CCR4 is known to be expressed on lymphocytes (e.g. Th2-cells andTreg-cells) titration curves of 9E10J and KM3060var on isolated PBLsfrom buffy coats were performed.

The antibodies 9E10J and KM3060var were biotinylated following themanual of the EZ-link Maleimide-PEO Solid Phase Biotinylation Kit(Pierce, Thermo Fisher, P.O. Box 117 Rockford, Ill. 61105 U.S.A). Thebiotinylation did not significantly affect the antibodies' ability torecognize their targets, as judged by assessing the binding of thebiotinylated and non-biotinylated antibodies to CCRF-CEM cells followingthe protocols given in example 2 (data not shown).

Human PBLs were prepared from fresh donor blood by Ficoll-(Hypaque)gradient centrifugation, washed in RPMI-1640/10% FCS. Leucosep tubes (50ml; Greiner Bio-One, Frikenhausen, Germany) were filled with 15 ml oflymphoprep (Fresenius Kabi Norge, Oslo, Norway) and centrifuged for 30sec at 1000 g at room temperature.

The buffy coat was diluted with 280 ml of 1× Dulbeccos PBS (pH 7.4) and35 ml of the diluted blood were transferred into each Leucosep tube (50ml; Greiner; Bio-One, Frikenhausen). Upon centrifugation for 10 min atroom temperature at 1000 g (no break, no acceleration), the target cellswere transferred into 50 ml tubes (50 ml Falcon, Greiner Bio-One,Frikenhausen, Germany), in a total volume of 50 ml of RPMI-medium. PBLswere pelleted for 15 min at 200 g at room temperature, resuspended in 50ml of RPMI-medium and filtered through a 70 μm cell strainer beforecounting cells. PBLs were diluted to a final cell number of 1.5×106cells/ml in 1×PBS (pH 7.4), supplemented with 0.2% BSA and 0.09% NaN3.

Biotinylated IgGs 9E10J and KM3060var were diluted from 20 μg/ml down to0.3 ng/ml in 1×PBS Dulbeccos (pH 7.4), supplemented with 0.2% BSA and0.09% NaN3 and incubated for 2 h on a total of 1.5×105 PBLs. Unbound IgGwas removed after washing cells twice with 150 μl of PBS (pH 7.4),supplemented with 0.2% BSA and 0.09% NaN3. Detection of bound IgG wasafter incubation with PE-labeled streptavidin (BD-Biosciences Norge;dilution of 1:100 in PBS (pH 7.4), supplemented with 0.2% BSA and 0.09%NaN3).

Cells were double stained with an anti-CD3-APC-conjugated antibody andwith an anti-CD8-PE-Cy5-conjugated antibody (BD-Biosciences Norge;dilution of 1:10 for both in PBS (pH 7.4), supplemented with 0.2% BSAand 0.09% NaN3). To compensate signal overlapping of the fluorochromes,an anti-CD4-PE-conjugated IgG (BD-Biosciences Norge) was added in aseparate well. Samples were analyzed on a FACS-cantoll (BD-BiosciencesNorge).

To assess binding to PBLs of 9E10J and KM3060var IgGs, lymphocytes werefirst gated out of the total events. Next, CD3-positive cells were gatedform lymphocyte population. The CD3-positive population was next dividedinto CD8-positive (=CD4-negative) and CD8-negative (=CD4-positive)cells, which include the Treg fraction. The signal intensity in thePE-channel indicates the binding of the 9E10J and KM3060var IgGs toCD8-negative (=CD4-positive) cells. Binding was expressed in % (positiveevents) after comparison to the negative control (=PE-conjugatedstreptavidin).

As presented in FIG. 30, 9E10J binds to gated CD4-positive cells(determined by CD8-negative staining). Binding is still detectable at aconcentration as low as 32 ng/ml (corresponding to ˜210 pM of IgG). Atthis concentration, no binding signal for comparator KM3060var wasdetectable. In this sense, 9E10J could be useful in immunotherapy forthe depletion of Treg-cells.

TABLE 1 SEQ ID NO: scFv 17G 25 Heavy QVQLVESGGGLVQPGGSLRLSCAASGFTFSchain FR1: 1 CDR 1: SYAMS 26 FR 2 WVRQAPGKGLEWVS 2 CDR 2:AISGSGGSTYYADSVKG 27 FR 3: RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAN 3 CDR 3:IRYSAGY 28 FR 4: WGQGTLVTVSS 29 Linker: KLSGSASAPKLEEGEFSEARV 30 LightSYVLTQPPSASGTPGQRVTISC chain FR1: 4 CDR 1: SGSSSNIGSNYVY 31 FR 2WYQQLPGTAPKLLIY 5 CDR 2: RNNQRPS 32 FR 3:GVPDRFSGSKSGTSASLAISGLRSEDEADYYC 6 CDR 3: AAWDDRLSGWV 33 FR 4:FGGGTKLTVL 34 scFv caggtgcagctggtggagtctgggggaggcttggtacag 17Gcctggggggtccctgagactctcctgtgcagcctctgg n.a.attcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaatattaggtatagtgcaggctactggggccagggaaccctggtcaccgtctcctcaaagctttcagggagtgcatccgccccaaaacttgaagaaggtgaattttcagaagcacgcgtatcctatgtgctgacacagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgaccgcctgagtggttgggtgttcggcggagggaccaagctgaccgtcct a 35 scFvQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ 17GAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT a.a.LYLQMNSLRAEDTAVYYCANIRYSAGYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDRLS GWVFGGGTKLTVL 69 V_(H (aa))QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANIRYSAGYWGQGTLVTVSS 70 V_(L (aa))SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISG LRSEDEADYYCAAWDDRLSGWVFGGGTKLTVL77 VH CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAG domainCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA (nt)TTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAATATTAGGTATAGTGCAGGCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 78 VLTCCTATGTGCTGACACAGCCACCCTCAGCGTCTGGGACC domainCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGC (nt)TCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACCGCCTGAGTGGTTGGGTGTTCGGCGGAGGG ACCAAGCTGACCGTCCTA

TABLE 2 SEQ ID NO: scFv 9E 36 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFS chainFR1: 7 CDR 1: NYAIS 37 FR 2 WVRQAPGQGLEWMG 8 CDR 2: GIIPIFGTANYAQKFQG 38FR 3: RVTITADESTSTAYMELSSLRSEDTAVYYCAR 9 CDR 3: RGGSYFDY 39 FR 4:WGQGTLVTVSS 40 Linker: KLSGSASAPKLEEGEFSEARV 41 LightSYVLTQPPSASGTPGQSVTISC chain FR1: 10 CDR 1: SGSTSNIGSHYVF 42 FR 2WYQQLPGTAPRLLIY 11 CDR 2: RNHQRPS 43 FR 3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC 12 CDR 3: AVWDDTLSGWV 44 FR 4:FGGGTKLTVL 45 scFv 9E caggtccagcttgtacagtctggggctgaggtgaagaag n.a.cctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcaactatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacgcggtgggagctactttgactactggggccagggaaccctggtcaccgtctcttcaaagctttcagggagtgcatccgccccaaaacttgaagaaggtgaattttcagaagcacgcgtatcctatgtgctgactcagccaccctcagcgtctgggacccccgggcagagcgtcaccatctcttgttctggaagcacctccaacatcggaagtcattatgtgttctggtaccagcagctcccaggaacggcccccagactcctcatctataggaatcatcagcggccctcaggggtccctgaccgactctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagtgtgggatgacaccctgagtggctgggtgttcggcggagggaccaagctgaccgtcctagcggccgctggatccgaacaaaagctgatctcagaagaagacctaaactcacatcaccatcaccatcac 46 scFv 9EQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQ a.a.APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDDTLSGWVFGGGTKLTVLAAAGSEQKLISEEDLNSHHHHHH 71 V_(H (aa))QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSS 72 V_(L (aa))SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISG LRSEDEADYYCAVWDDTLSGWVFGGGTKLTVL79 VH CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAG domainCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGA (nt)GGCACCTTCAGCAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA 80 VLTCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACC domainCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACC (nt)TCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGG ACCAAGCTGACCGTCCTA

TABLE 3 SEQ ID NO: scFv 1O 47 Heavy QVQLVESGGGLVQPGRSLRLSCAASGFTFS chainFR1: 1 or CDR 1: SYAMS 13* 48 FR 2 WVRQAPGKGLEWVS 2 or CDR 2:AISGSGGSTYYADSVKG 14** 49 FR 3: RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 15 CDR3: GAGEGRGLGVV 50 FR 4: MGQGTLVTVSS 51 Linker: KLSGSASAPKLEEGEFSEARV 52Light QAVVTQEPSLTVSPGGTVTLTC chain FR1: 16 CDR 1: ASSTGAVTSGYFPN 53 FR 2WFQQKPGQAPRALIY 17 CDR 2: STTNKHS 54 FR 3:WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC 18 CDR 3: LLYYGGARV 55 FR 4: FGGGTKLTVL56 scFV 1O caggtgcagctggtggaatctgggggaggcttggtac n.a.agcctggcaggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaagggggccggcgagggtcgagggcttggagtggttatgggccagggaaccctggtcaccgtctcctcaaagctttcagggagtgcatccgccccaaaacttgaagaaggtgaattttcagaagcacgcgtacaggctgtggtgacccaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtgcttccagcactggagcagtcaccagtggttactttccaaactggttccagcagaaacctggacaagcacccagggcactcatttatagtacaaccaacaaacactcctggacccctgcccggttctcaggctccctccttgggggcaaagctgccctgacactgtcaggtgtgcagcctgaggacgaggctgagtattactgcctgctctactatggtggtgctcgggtgttcggcggagggaccaagctcaccgtcctagcggccgctggatccgaacaaaagctgatctcagaagaagacctaaactcacatcaccatcacc atcac 57 scFv 1OQVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWV a.a.RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGEGRGLGVVMGQGTLVTVSSKLSGSASAPKLEEGEFSEARVQAVVTQEPSLTVSPGGTVTLTCASSTGAVTSGYFPNWFQQKPGQAPRALIYSTTNKHSWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCLLYYGGARVFGGGTKLTVLAAAGSEQKL ISEEDLNSHHHHHH 73 V_(H (aa))QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGEGRGLGVVMG QGTLVTVSS 74 V_(L (aa))QAVVTQEPSLTVSPGGTVTLTCASSTGAVTSGYFPNWFQQKPGQAPRALIYSTTNKHSWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCLLYYGGARVFGGGTKLTVL 81 VHCAGGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTAC domainAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTC (nt)TGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAGGGGGCCGGCGAGGGTCGAGGGCTTGGAGTGGTTATGGGC CAGGGAACCCTGGTCACCGTCTCCTCA 82 VLCAGGCTGTGGTGACCCAGGAGCCCTCACTGACTGTGT domainCCCCAGGAGGGACAGTCACTCTCACCTGTGCTTCCAG (nt)CACTGGAGCAGTCACCAGTGGTTACTTTCCAAACTGGTTCCAGCAGAAACCTGGACAAGCACCCAGGGCACTCATTTATAGTACAACCAACAAACACTCCTGGACCCCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACACTGTCAGGTGTGCAGCCTGAGGACGAGGCTGAGTATTACTGCCTGCTCTACTATGGTGGTGCTCGGGT GTTCGGCGGAGGGACCAAGCTCACCGTCCTA*SEQ ID NO: 1 is identical to SEQ ID NO: 13 **SEQ ID NO: 2 is identicalto SEQ ID NO: 14

TABLE 4 SEQ ID NO: scFv 11F 58 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTchain FR1: 19 CDR 1: GYYMH 59 FR 2 WVRQAPGQGLEWMG 20 CDR 2:WINPNSGGTNYAQKFQG 60 FR 3: RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 21 CDR 3:GTVTRIQGRSLYGMDV 61 FR 4: WGQGTTVTVSS 62 Linker: KLSGSASAPKLEEGEFSEARV63 Light ETTLTQSPGTLSLSPGEGVTLSC chain FR1: 22 CDR 1: RASQSVNRRYLA 64 FR2 WYQQKPGQAPRLLIY 23 CDR 2: GASSRAT 65 FR 3:GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 24 CDR 3: QQYGSSPLT 66 FR 4: FGGGTKVEIK67 scFv 11F caggtccagcttgtgcagtctggggctgaggtgaaga n.a.agcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagaggtacggtgactaggattcagggccgctctctctacggtatggacgtctggggccaagggaccacggtcaccgtctcttcaaagctttcagggagtgcatccgccccaaaacttgaagaaggtgaattttcagaagcacgcgtagaaacgacactcacgcagtctccaggcaccctgtctttgtctccaggggaaggagtcaccctctcctgcagggccagtcagagtgttaacaggaggtacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggggcatccagcagggccactggcatccctgacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcacccctcactttcggcggagggaccaaggtggaaatcaaagcggccgctggatccgaacaaaagctgatctcagaagaagacctaaactcac atcaccatcaccatcac 68 scFv 11FQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV a.aRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGTVTRIQGRSLYGMDVWGQGTTVTVSSKLSGSASAPKLEEGEFSEARVETTLTQSPGTLSLSPGEGVTLSCRASQSVNRRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKAAAGS EQKLISEEDLNSHHHHHH 75 V_(H (aa))QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGTVTRIQGRSLYG MDVWGQGTTVTVSS 76 V_(L (aa))ETTLTQSPGTLSLSPGEGVTLSCRASQSVNRRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK83 VH CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGA domainAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTC (nt)TGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGGTACGGTGACTAGGATTCAGGGCCGCTCTCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CTTCA 84 VLGAAACGACACTCACGCAGTCTCCAGGCACCCTGTCTT domainTGTCTCCAGGGGAAGGAGTCACCCTCTCCTGCAGGGC (nt)CAGTCAGAGTGTTAACAGGAGGTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGGGCATCCAGCAGGGCCACTGGCATCCCTGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCCTCACTTT CGGCGGAGGGACCAAGGTGGAAATCAAA

TABLE 5 IgG sequences of 17G SEQ ID NO: IgG1caggtgcagctggtggagtctgggggaggcttggtacagcctg 85 heavygggggtccctgagactctcctgtgcagcctctggattcacctt chaintagcagctatgccatgagctgggtccgccaggctccagggaag (nt)gggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaatattaggtatagtgcaggctactggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg gtaaa SEQ ID NO: Lambdatcctatgtgctgacacagccaccctcagcgtctgggaccccc 86 lightgggcagagggtcaccatctcttgttctggaagcagctccaac chainatcggaagtaattatgtatactggtaccagcagctcccagga (nt)acggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgaccgcctgagtggttgggtgttcggcggagggaccaagctgaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtg gcccctacagaatgttca SEQ ID NO:IgG1 qvqlvesggglvqpggslrlscaasgftfssyamswvrqapg 87 heavykglewvsaisgsggstyyadsvkgrftisrdnskntlylqmn chainslraedtavyycanirysagywgqgtlvtvssastkgpsvfp (aa)lapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgn vfscsvmhealhnhytqkslslspgkSEQ ID NO: Lambda syvltqppsasgtpgqrvtiscsgsssnigsnyvywyqqlpg 88 lighttapklliyrnngrpsgvpdrfsgsksgtsaslaisglrsede chainadyycaawddrlsgwvfgggtkltvlgqpkaapsvtlfppss (aa)eelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektv aptecs

TABLE 6 IgG sequences of 9E SEQ IgG1caggtccagcttgtacagtctggggctgaggtgaagaagc ID heavyctgggtcctcggtgaaggtctcctgcaaggcttctggagg NO: chaincaccttcagcaactatgctatcagctgggtgcgacaggcc 89 (nt)cctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacgcggtgggagctactttgactactggggccagggaaccctggtcaccgtctcttcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagc ctctccctgtctccgggtaaa SEQLambda tcctatgtgctgactcagccaccctcagcgtctgggaccc ID lightccgggcagagcgtcaccatctcttgttctggaagcacctc NO: chaincaacatcggaagtcattatgtgttctggtaccagcagctc 90 (nt)ccaggaacggcccccagactcctcatctataggaatcatcagcggccctcaggggtccctgaccgactctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagtgtgggatgacaccctgagtggctgggtgttcggcggagggaccaagctgaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacag aatgttca SEQ IgG1qvqlvqsgaevkkpgssvkvsckasggtfsnyaiswvrqa ID heavypgqglewmggiipifgtanyaqkfqgrvtitadeststay NO: chainmelsslrsedtavyycarrggsyfdywgqgtlvtvssast 91 (aa)kgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqks lslspgk SEQ Lambdasyvltqppsasgtpgqsvtiscsgstsnigshyvfwyqql ID lightpgtaprlliyrnhqrpsgvpdrlsgsksgtsaslaisglr NO: chainsedeadyycavwddtlsgwvfgggtkltvlgqpkaapsvt 92 (aa)lfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvt hegstvektvaptecs

TABLE 7 IgG sequences of 10 SEQ IgG1caggtgcagctggtggaatctgggggaggcttggtacagc ID heavyctggcaggtccctgagactctcctgtgcagcctctggatt NO: chaincacctttagcagctatgccatgagctgggtccgccaggct 93 (nt)ccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaagggggccggcgagggtcgagggcttggagtggttatgggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg cagaagagcctctccctgtctccgggtaaaSEQ Lambda caggctgtggtgacccaggagccctcactgactgtgtccc ID lightcaggagggacagtcactctcacctgtgcttccagcactgg NO: chainagcagtcaccagtggttactttccaaactggttccagcag 94 (nt)aaacctggacaagcacccagggcactcatttatagtacaaccaacaaacactcctggacccctgcccggttctcaggctccctccttgggggcaaagctgccctgacactgtcaggtgtgcagcctgaggacgaggctgagtattactgcctgctctactatggtggtgctcgggtgttcggcggagggaccaagctcaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaat gttca SEQ IgG1qvqlvesggglvqpgrslrlscaasgftfssyamswvrqa ID heavypgkglewvsaisgsggstyyadsvkgrftisrdnskntly NO: chainlqmnslraedtavyycakgagegrglgvvmgqgtlvtvss 95 (aa)astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskitvdksrwqqgnvfscsvmhealhnhyt qkslslspgk SEQ Lambdaqavvtqepsltvspggtvtltcasstgavtsgyfpnwfqq ID lightkpgqapraliysttnkhswtparfsgsllggkaaltlsgv NO: chainqpedeaeyycllyyggarvfgggtkltvlgqpkaapsvtl 96 (aa)fppsseelqankatlvclisdfypgavtvawkadsspvkagvetftpskqsnnkyaassylsltpeqwkshrsyscqvth egstvektvaptecs

TABLE 8 IgG sequences of 11F SEQ IgG1caggtccagcttgtgcagtctggggctgaggtgaagaagc ID heavyctggggcctcagtgaaggtctcctgcaaggcttctggata NO: chaincaccttcaccggctactatatgcactgggtgcgacaggcc 97 (nt)cctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagaggtacggtgactaggattcagggccgctctctctacggtatggacgtctggggccaagggaccacggtcaccgtctcttcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg gtaaa SEQ Kappagaaacgacactcacgcagtctccaggcaccctgtctttgt ID lightctccaggggaaggagtcaccctctcctgcagggccagtca NO: chaingagtgttaacaggaggtacttagcctggtaccagcagaaa 98 (nt)cctggccaggctcccaggctcctcatctatggggcatccagcagggccactggcatccctgacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcacccctcactttcggcggagggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt SEQ IgG1qvqlvqsgaevkkpgasvkvsckasgytftgyymhwvrqa ID heavypgqglewmgwinpnsggtnyaqkfqgrvtmtrdtsistay NO: chainmelsrlrsddtavyycargtvtriqgrslygmdvwgqgtt 99 (aa)vtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheal hnhytqkslslspgk SEQ Kappaettltqspgtlslspgegvtlscrasqsvnrrylawyqqk ID lightpgqaprlliygassratgipdrfsgsgsgtdftltisrle NO: chainpedfavyycqqygsspltfgggtkveikrtvaapsvfifp 100 (aa)psdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthq glsspvtksfnrgec

TABLE 9 CCR4⁺ CCR4⁺ CCR4⁺ IgG DT40 DT40 (2) HEK293T CCRF-CEM 17G 0.42 ±0.07 nM 0.42 ± 0.03 nM 0.62 ± 0.22 nM 0.16 ± 0.02 nM 9E 0.72 ± 0.28 nM0.17 ± 0.03 nM 0.36 ± 0.19 nM 0.39 ± 0.02 nM KM3060var 3.67 ± 0.29 nM4.82 ± 0.16 nM 11.40 ± 1.74 nM  79.83 ± 6.59 nM 

TABLE 10 SEQ ID NOS scFv KM3060var 141 HeavyEVQLVESGGDLMKPGGSLKISCAASGFIFS chain FR1: 142 CDR 1: NYGMS 143 FR 2WVRQTPDMRLEWVA 144 CDR 2: TISSASTYSYYPDSVKG 145 FR 3:RFTISRDNAENSLYLQMNSLRSEDTGIYYC 146 CDR 3: GRHSDGNFAFGY 147 FR 4:WGRGTLVTVSS 148 Linker: KLSGSASAPKLEEGEFSEARV 149 LightDVLMTQTPLSLPVSLGDQASISC chain FR1: 150 CDR 1: RSSRNIVHINGDTYLE 151 FR 2WYLQRPGQSPKLLIY 152 CDR 2: KVSNRFS 153 FR 3:GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC 154 CDR 3: FQGSLLPWT 155 FR 4:FGGGTRLEIK 156 scFv gaggtgcagctggtcgagagcggaggcgacctgatgaagcctggcggKM3060 cagcctgaagatcagctgcgccgccagcggcttcatcttcagcaact varacggcatgagctgggtgcgccagacccccgacatgcggctggaatgg n.a.gtggcaaccatcagcagcgccagcacctacagctactaccccgacagcgtgaagggccggttcaccatcagccgggacaacgccgagaacagcctgtatctgcagatgaacagcctgcggagcgaggacaccggcatctactactgcggcagacacagcgacggcaacttcgccttcggctactggggcagaggcaccctggtgaccgtgtccagcaagctttccggcagcgcctccgcccccaagctggaagagggcgagttcagcgaggcacgcgtggacgtgctgatgacccagacccctctgagcctgcccgtgtccctgggcgaccaggccagcatcagctgcagaagcagccggaacatcgtccacatcaacggcgacacctacctggaatggtatctgcagcggcctggacagagccccaagctgctgatctacaaggtgtccaaccggttcagcggcgtgcccgacagattcagcggaagcggctccggcaccgacttcaccctgaagatctcccgggtggaggccgaggacctgggcgtgtactactgctttcaaggcagcctgctgccctggaccttcggcggaggcacacggctggaaatcaaagcggccgctggatccgaacaaaagctgatctcagaagaagacctaaactcacatcaccatcaccatcac 157 scFv-EVQLVESGGDLMKPGGSLKISCAASGFIFSNYGMSWVRQTPDMRLEW KM3060VATISSASTYSYYPDSVKGRFTISRDNAENSLYLQMNSLRSEDTGIY varYCGRHSDGNFAFGYWGRGTLVTVSS KLSGSASAPKLEEGEFSEARV D a.a.VLMTQTPLSLPVSLGDQASISCRSSRNIVHINGDTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSLLPWTFGGGTRLEIKAAAGSEQKLISEEDLNSHHHHHH

TABLE 11 SEQ ID NO: scFv 9E10J 36 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1: 101 CDR 1: SYAIS 37 FR 2 WVRQAPGQGLEWMG 8 CDR 2:GIIPIFGTANYAQKFQG 102 FR 3: RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 9 CDR 3:RGGSYFDY 39 FR 4: WGQGTLVTVSS 40 Link- KLSGSASAPKLEEGEFSEARV er: 41Light SYVLTQPPSASGTPGQSVTISC chain FR1: 10 CDR 1: SGSTSNIGSHYVF 42 FR 2WYQQLPGTAPRLLIY 11 CDR 2: RNHQRPS 43 FR 3:GVPDRLSGSKSGTSASLAISGLRSEDEADYYC 12 CDR 3: AVWDDTLSGWV 44 FR 4:FGGGTKLTVL 103 scFv CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGC 9E10JCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGC GGAGGGACCAAGCTGACCGTCCTA 104scFv QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 9E10JPGQGLEWMGGIIPIFGTANYAQKFQGRVTMTRDTSTSTVY a.a.MELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVSYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAVWDDTLSGWVFG GGTKLTVL 105 V_(H(aa))QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSS 72 V_(L(aa))SYVLTQPPSASGTPGQSVTISCSGSTSNIGSHYVFWYQQLPGTAPRLLIYRNHQRPSGVPDRLSGSKSGTSASLAISGLR SEDEADYYCAVWDDTLSGWVFGGGTKLTVL106 V_(H) CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGC DomainCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGG (nt)CACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTG GGGCCAAGGGACCCTGGTCACCGTCTCCTCA80 V_(L) TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCC DomainCCGGGCAGAGCGTCACCATCTCTTGTTCTGGAAGCACCTC (nt)CAACATCGGAAGTCATTATGTGTTCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAGACTCCTCATCTATAGGAATCATCAGCGGCCCTCAGGGGTCCCTGACCGACTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGTGTGGGATGACACCCTGAGTGGCTGGGTGTTCGGCGGAGGGACCAAGCT GACCGTCCTA

TABLE 12 SEQ ID NO: scFv 9E1D 36 Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSchain FR1: 7 CDR1: NYAIS 37 FR2 WVRQAPGQGLEWMG 8 CDR2: GIIPIFGTANYAQKFQG38 FR3: RVTITADESTSTAYMELSSLRSEDTAVYYCAR 9 CDR3: RGGSYFDY 39 FR4:WGQGTLVTVSS 40 Linker: KLSGSASAPKLEEGEFSEARV 107 LightQPVLTQPPSASGTPGQRVTISC chainFR1: 108 CDR1: SGGGSNIGRRFVN 109 FR2WYQQLPGTAPKLLIY 110 CDR2: RNNQRPS 111 FR3:GVPDRFSGSKSGTSASLVISGLRSEDEADYYC 112 CDR3: AAWDDSLSGWV 44 FR4:FGGGTKLTVL 113 scFv9E1D CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCAAAGCTTTCAGGGAGTGCATCCGCCCCAAAACTTGAAGAAGGTGAATTTTCAGAAGCACGCGTACAGCCTGTGCTGACTCAGCCCCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGCGGATCCAACATCGGAAGAAGGTTTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTC CTA 114 scFv9E1DQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQ a.a.APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSSKLSGSASAPKLEEGEFSEARVQPVLTQPPSASGTPGQRVTISCSGGGSNIGRRFVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLVISGLRSEDEADYYCAAWDDSL SGWVFGGGTKLTVL 71 V_(H(aa))QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTVSS 115 V_(L(aa))QPVLTQPPSASGTPGQRVTISCSGGGSNIGRRFVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLVISG LRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL79 V_(H)domain CAGGTCCAGCTTGTACAGTCTGGGGCTGAGGTGAAGAAG (nt)CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAACTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACGCGGTGGGAGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA 116 V_(L)domainCAGCCCCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTC (nt)ACCATCTCTTGTTCTGGAGGCGGATCCAACATCGGAAGAAGGTTTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTC CTA

TABLE 13 Sequences of 9E10J IgG. SEQ. ID NO: IgG1caggtccagcttgtacagtctggggctgaggtgaagaagcct 117 heavygggtcctcggtgaaggtctcctgcaaggcttctggaggcacc chainttcagcagctatgctatcagctgggtgcgacaggcccctgga (nt)caagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcaccatgaccagggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacgcggtgggagctactttgactactggggccaagggaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagetgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ. ID NO: Lambdatcctatgtgctgactcagccaccctcagcgtctgggaccccc 118 Lightgggcagagcgtcaccatctcttgttctggaagcacctccaac chainatcggaagtcattatgtgttctggtaccagcagctcccagga (nt)acggcccccagactcctcatctataggaatcatcagcggccctcaggggtccctgaccgactctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagtgtgggatgacaccctgagtggctgggtgttcggcggagggaccaagctgaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaecacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtg gcccctacagaatgttca SEQ. IDNO: IgG- qvqlvqsgaevkkpgssvkvsckasggtfssyaiswvrqapg 119 heavyqglewmggiipifgtanyaqkfqgrvtmtrdtststvymels chainslrsedtavyycarrggsyfdywgqgtlvtvssastkgpsvf (aa)plapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg nvfscsvmhealhnhytqkslslspgkSEQ. ID NO: Lambda syvltqppsasgtpgqsvtiscsgstsnigshyvfwyqqlpg 120 Light-taprlliyrnhqrpsgvpdrlsgsksgtsaslaisglrsede chainadyycavwddtlsgwvfgggtkltvlgqpkaapsvtlfppss (aa)eelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektv aptecs

TABLE 14 Sequences of 9E1D IgG. SEQ. ID NO: IgG1caggtccagcttgtacagtctggggctgaggtgaagaagcct 121 heavygggtcctcggtgaaggtctcctgcaaggcttctggaggcacc chainttcagcaactatgctatcagctgggtgcgacaggcccctgga (nt)caagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagacgcggtgggagctactttgactactggggccagggaaccctggtcaccgtctcttcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ. ID NO: Lambdacagcctgtgctgactcagcccccctcagcgtctgggaccccc 122 Light-gggcagagggtcaccatctcttgttctggaggcggatccaac chainatcggaagaaggtttgtaaactggtaccagcagctcccagga (nt)acggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggtcatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcctgagtggttgggtgttcggcggagggaccaagctgaccgtcctaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtg gcccctacagaatgttca SEQ. IDNO: IgG- qvqlvqsgaevkkpgssvkvsckasggtfsnyaiswvrqapg 123 heavyqglewmggiipifgtanyaqkfqgrvtitadeststaymels chainslrsedtavyycarrggsyfdywgqgtlvtvssastkgpsvf (aa)plapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqg nvfscsvmhealhnhytqkslslspgkSEQ. ID NO: Lambda qpvltqppsasgtpgqrvtiscsgggsnigrrfvnwyqqlpg 124 Light-tapklliyrnnqrpsgvpdrfsgsksgtsaslvisglrsede chainadyycaawddslsgwvfgggtkltvlgqpkaapsvtlfppss (aa)eelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylsltpeqwkshrsyscqvthegstvektv aptecs

TABLE 15 Apparent affinities (IC₅₀ and K_(D)) as determined from bindinginhibition experiments DT-40-CCR4⁺ DT-40-CCR4⁺ CCRF-CEM (bio-IgG 9E(bio-IgG 9E (bio-IgG 9E 3.3 nM) 0.7 nM) 0.7 nM) IC₅₀ K_(D) IC₅₀ K_(D)IC₅₀ K_(D) IgG (nM) (nM) (nM) (nM) (nM) (nM) 9E 3.50 2.1 0.44 0.39 0.380.33 9E10J 0.15 0.09 0.06 0.05 0.07 0.06

TABLE 16 Calculated antibody concentrations leading to 50% TARC-inducedCCR4 signalling (IC₅₀), as determined in Ca⁺⁺ flux assays IC₅₀ (ng/ml)Conc. range: Conc. range: Conc. range: Antibody (IgG1) 1-100 ng/ml0.1-10 μg/ml 3.2 ng/ml-10 μg/ml 9E 21.36 ng/ml 46.15 ng/ml ND (142.4 pM)(307.7 pM) 9E10J 7.24 ng/ml 7.88 ng/ml 6.48 ng/ml (48.3 pM) (52.5 pM)(43.3 pM)

TABLE 17 Comparative ADCC activity of anti-CCR4 antibodies. 9E 9E10JKM3060var Max Max Max EC₅₀ killing EC₅₀ killing EC₅₀ killing (ng/mL) (%)(ng/mL) (%) (ng/mL) (%) Mean 185.6 31.8 24.7 42.4 367.0 38.8 SD 199.42.3 15.9 15.6 549.2 9.9 N 2 2 5 5 3 3

TABLE 18 Comparative ADCC activity of defucosylated 9E10J-anti-CCR4antibody in comparison to unmodified 9E10J and comparator KM3060var.9E10J- 9E10J defucosylated KM3060var Max Max Max EC₅₀ killing EC₅₀killing EC₅₀ killing (ng/mL) (%) (ng/mL) (%) (ng/mL) (%) Mean 8.7 21.42.1 78.3 405 40.4 SD 0.4 16.9 0.83 13.5 466 17.5 N 2 2 4 4 3 3

TABLE 19 Apparent affinities (K_(D)) as determined by saturated antibodytitration on cells in flow cytometry. scFv DT-40 CCR4⁺ IgG CCRF-CEM 9E4.95 ± 2.67 nM 9E 0.37 ± 0.11 nM 9E1D 5.22 ± 1.61 nM 9E1D ND 9E10J 1.17± 0.45 nM 9E10J 0.13 ± 0.03 nM ND, not determined;

TABLE 20 IC₅₀ values and calculated affinities (K_(D)) for cell-bindinginhibition of biotinylated MDC, as determined from competition bindingexperiments on DT-40-CCR4⁺ and CCRF-CEM cells. DT-40-CCR4⁺ cellsCCRF-CEM cells Inhibitor IC₅₀ (nM) K_(D) (nM) IC₅₀ (nM) K_(D) (nM) IgG9E10J 0.76 0.01 11.21 0.20 6.28 39.97 MDC 10.76 0.19 10.10 0.18 10.1269.36

TABLE 21 IC₅₀ values for cell-binding inhibition of biotinylated TARC,as determined from competition binding experiments on DT-40-CCR4⁺ andCCRF-CEM cells. IC₅₀ (nM) Inhibitor DT-40-CCR4⁺ cells CCRF-CEM cells IgG9E10J 0.26 4.26 0.40 4.57 TARC 40.93 43.44 34.58 31.79

TABLE 22 Overview of determined IC₅₀-values from competitionexperiments. IC₅₀ (nM) 9E10J 9E MDC 0.85 3.9 TARC 2.5 12.5

TABLE 23 Consensus sequences CDR/variable SEQ X = any amino acid chainID NO: XYAXS (X₁YAX₂S) Heavy CDR1 125 S/N Y A I/M S Heavy CDR1 126XIXPXXGXXNYAQKFQG Heavy CDR2 127 G/A I I/S P I/S F/G G T/S A/T NYAQKFQGHeavy CDR2 128 SGXXSNIGXXXVX Light CDR1 129 SG S/G T/G SNIG S/R R/H Y/FV Y/F Light CDR1 130 RNXQRPS Light CDR2 131 RN H/N QRPS Light CDR2 132AXWDDXLSGWY Light CDR3 133 A A/V WDD S/T LSGWY Light CDR3 134 XYAISheavy CDR1 135 S/N YAIS heavy CDR1 136 AXWDDXLSGWV Light CDR3* 137 A A/VWDD S/T LSGWV Light CDR3* 138 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSXYAISWVR VH139 QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTXTXDXSTSTXYMELSSLRSEDTAVYYCARRGGSYFDYWGQGTLVTV SSXXVLTQPPSASGTPGQXVTISCSGXXSNIGXXXVXWYQQ VL 140LPGTAPXLLIYRNXQRPSGVPDRXSGSKSGTSASLXISGL RSEDEADYYCAXWDDXLSGWVFGGGTKLTVL*corrected sequence

TABLE 24 % aggregation IgGs alone 9E10J Non detectable Anti-GFP Nondetectable ligands alone TARC 98% MDC 97% Preincubation IgGs/ligandsanti-GFP/TARC 88% anti-GFP/MDC 100%  9E10J/TARC 82% 9E10J/MDC 93%Mixture IgG/ligands 9E10J/TARC 100%  9E10J/MDC 78%

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A human antibody which binds to an epitope in the extracellulardomain of human CC chemokine receptor 4 (CCR4) and which is capable ofinhibiting the binding of macrophage-derived chemokine (MDC) and/orthymus and activation regulated chemokine (TARC) to CCR4, wherein saidhuman antibody can compete for binding to CCR4 with an antibody thatcomprises (a) variable heavy (VH) CDR1 of SEQ ID NO:101; (b) VH CDR2 ofSEQ ID NO:8; (c) VH CDR3 of SEQ ID NO:9; (d) variable light (VL) CDR1 ofSEQ ID NO:10; (e) VL CDR2 of SEQ ID NO:11; and (f) VL CDR3 of SEQ IDNO:12.
 2. The human antibody of claim 1, wherein said human antibodycomprises (i) VH CDR1 of SEQ ID NO: 126; (ii) VH CDR2 of SEQ ID NO: 128;(iii) VH CDR3 of SEQ ID NO: 9; (iv) VL CDR1 of SEQ ID NO: 130; (v) VLCDR2 of SEQ ID NO: 132; and (vi) VL CDR3 of SEQ ID NO:
 138. 3. The humanantibody of claim 1, wherein said human antibody comprises a VH domainof SEQ ID NO: 139 and/or a VL domain of SEQ ID NO:
 140. 4. The antibodyof claim 1, wherein said human antibody can bind to substantially thesame epitope as the antibody that comprises (a) variable heavy (VH) CDR1of SEQ ID NO:101; (b) VH CDR2 of SEQ ID NO:8; (c) VH CDR3 of SEQ IDNO:9; (d) variable light (VL) CDR1 of SEQ ID NO:10; (e) VL CDR2 of SEQID NO:11; and (f) VL CDR3 of SEQ ID NO:12.
 5. The human antibody ofclaim 1, wherein said antibody is a fully human antibody.
 6. The humanantibody of claim 1, wherein said human antibody comprises all or aportion of an antibody heavy chain constant region and/or an antibodylight chain constant region.
 7. The human antibody of claim 1, whereinsaid human antibody is an IgG antibody.
 8. The human antibody of claim1, wherein at least 10, 20, 30, 40 50, 60, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or at least 99% of the total complexN-glycoside-linked sugar chains bound to the Fc region of said humanantibody are sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain.
 9. The humanantibody of claim 1, wherein said human antibody is an antigen bindingfragment of an antibody.
 10. The human antibody of claim 1, wherein saidhuman antibody is an antigen binding fragment of an antibody and whereinsaid antigen binding fragment is a Fab′, Fab, F(ab′)₂, single domainantibody, T and Abs dimer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibody,minibody, diabody, bispecific antibody fragment, bibody, tribody,sc-diabody, kappa(lamda) body, bispecific T-cell engager (BiTE), dualvariable domain immunoglobulin (DVD-Ig), small immunoprotein (SIP),small modular immunopharmaceutical (SMIP), dual affinity retargetingconstruct (DART) or a small antibody mimetic comprising the CDRs.
 11. Animmunoconjugate comprising the human antibody of claim 1 attached to atleast a therapeutic or diagnostic agent.
 12. An immunoconjugatecomprising the human antibody of claim 1 attached to at least atherapeutic or diagnostic agent selected from a radiotherapeutic agent,chemotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent,anti-tubulin drug, anti-cellular or cytotoxic agent, steroid, cytokineantagonist, cytokine expression inhibitor, chemokine antagonist,chemokine expression inhibitor, anti-inflammatory corticosteroid orNSAIDs, coagulant or anti-viral agent, wherein said anti-viral agent ispreferably selected from the group consisting of a nucleoside, anucleoside reverse transcriptase inhibitor, a non-nucleoside reversetranscriptase inhibitor and a protease inhibitor.
 13. An immunoconjugatecomprising the human antibody of claim 1 attached to daunorubicin,Doxorubicin, Cytarabine, 6-thioguanine, Mitoxantrone, busulfan(Myleran®), dasatinib (Sprycel™), prednisone, vincristine sulfate(Oncovin®), Chlorambucil, Fludarabine, Pentostatin or Cladribine.
 14. Acomposition comprising at least the human antibody according any claim1, wherein said composition is a pharmaceutically acceptablecomposition, a liposome, a nanoparticle composition, and/or furthercomprises at least a further therapeutic agent.